Increasing life span by modulation of Smek

ABSTRACT

The Smek (Suppressor of mek null) gene is described and characterized. Smek acts in the stress response pathway of animals by binding to and enhancing the transcription of FOXO, thereby providing the link between the stress response pathway and the insulin/IGF-1 pathway. Given the link between both the stress response pathway and the insulin/IGF-1 pathway and longevity, Smek1 represents an essential target for modulation of life span and the stress response. Methods of increasing life span and stress tolerance by modulation of Smek activity are disclosed, as are screening methods for identifying compounds that modulate Smek activity. In addition, recombinant animals expressing the Smek gene that have a longer life span and enhanced stress tolerance, and methods of using the Smek gene to modulate both longevity and stress tolerance, are described.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/165,819 filed Jun. 24, 2005 now U.S. Pat. No. 7,288,385, which claimsthe benefit of U.S. Provisional Application No. 60/583,284 filed Jun.25, 2004, both incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with United States government support underGrant No. RO1 CA082683, Grant No. 5 F32 DK060367, Grant No. CA054418,and Grant No. DK070696 from the National Institutes of Health. TheUnited States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The field of the invention relates to methods of modulating at least onetrait in an animal. Such traits include increased life span, enhancedstress resistance and other traits associated with the stress responsepathway. Also encompassed are transgenic animals produced by thedisclosed methods.

2. Description of the Related Art

Stress response is a physiological phenomenon universal to all livingspecies, which are constantly exposed to internal and externalenvironmental challenges. Naturally the ability of an organism to reactto various stress conditions plays a critical role in determining itschances of survival. One interesting example is the phenomenon oforganismal longevity, i.e. long-term survival of an organism, which isclosely associated stress resistance from species diverse as yeast andmammals (Guarente and Kenyon 2000; Kenyon 2001; Burgering and Kops 2002;Hekimi and Guarente 2003). Recent studies in model organisms, especiallyC. elegans, showed that the aging process is regulated by a conservedmechanism (Kenyon 2001). It has been well established that mutations inthe insulin/IGF-1 signaling pathway in worm leads to extended life span,which is dependent on Daf16, a homolog of the vertebrate forkheadtranscription factors (FOXOs) (Kenyon 2001). Among multiple processesperturbed in these long lived mutants, it is striking that stressresistance is the one that is most tightly coupled to longevity (Kenyon2001). This raises the possibility that signaling pathways mediatingstress response might play a direct role in life span extension, whichis supported by recent findings on the stress-dependent regulation ofFOXO by histone deacetylase SIRT1 (Brunet, Sweeney et al. 2004; Motta,Divecha et al. 2004).

Stress response pathways mediate cellular responses towards variousphysiological and environmental stress signals. Members of a family ofstress activated kinases, including JNK and p38 MAP kinases, play acentral role in stress response pathways (Chang and Karin 2001; Morrisonand Davis 2003). Only a few studies, however, have directly examined anddemonstrated a role for stress signaling proteins in the aging process(Wang, Bohmann et al. 2003). Indeed the molecular links that connectstress signaling to aging, and how signals from distinct pathways suchas stress response pathway and insulin/IGF-1 pathway may be integratedto specify life span, are poorly understood. Filling such a gap byunifying these two major signaling routes will not only advance ourunderstanding of the mechanisms of aging, but also provide insights intothe signaling network implicated in various human diseases includingcancer and diabetes. Thus there is a need for identification of themolecular links that connect the stress response to aging. Further,there is a need for methods of modulation of that molecular link toextend life span and increase stress tolerance of animals.

SUMMARY OF THE INVENTION

The present invention meets the above needs by providing the identity ofa key protein family that ties the stress signaling pathway with aging.Identification of this protein family, Suppressor of MEK null (Smek),has led to various aspects of the present invention as set forth belowincluding, without limitation, methods of increasing the lifespan ofanimals, methods of screening for compounds that increase the life spanof an animal and/or modulate the stress response of an animal, andtransgenic animals and cells that have a longer life span and/orenhanced resistance to stress.

One aspect of the present invention includes a method of increasing thelife span of animal by modulating the activity or expression of a Smekprotein. The method includes administering to the animal a compound thatmodulates the activity or expression of a Smek protein. In certainembodiments in higher organisms, a Smek protein may be either Smek1 orSmek2. In preferred examples of such embodiments, the compoundselectively modulates a Smek1 protein or a Smek2 protein, but not both.In yet other embodiments, the compound may decrease or preferablyincrease the activity or expression of the Smek protein of interest. Insome embodiments, the increase in activity or expression of a Smekprotein is due to enhanced transcription, enhanced translation, enhancedphosphorylation, or enhanced affinity for the FOXO transcription factor.In various embodiments, the animal may be a vertebrate animal, a mammal,or a human, pig, cow, sheep, horse, cat, dog, chicken, or turkey.

Another aspect of the present invention includes methods of increasingthe life span of an animal by administering to the animal atherapeutically effective amount of a Smek protein. In certainembodiments in higher organisms, the Smek protein may be either Smek1 orSmek2. In various embodiments, the animal may be a vertebrate animal, amammal, or a human, pig, cow, sheep, horse, cat, dog, chicken, orturkey.

Yet another aspect of the present invention is a method of identifying acompound that increases the lifespan of an animal. The method includes

-   -   contacting an isolated cell that expresses a Smek protein with a        compound;    -   detecting the activity or expression of the Smek protein; and    -   comparing the activity or expression of the Smek protein after        contacting and the activity or expression of the Smek protein in        the absence of the compound to determine whether the compound        increases the activity or expression of the Smek protein thereby        increasing lifespan.

In certain embodiments in higher organisms, the Smek protein may beeither Smek1 Smek2. In various embodiments, the animal may be avertebrate animal, a mammal, or a human, pig, cow, sheep, horse, cat,dog, chicken, or turkey. In various other embodiments, the isolated cellmay be a prokaryotic cell, a eukaryotic cell, a plant cell, a vertebrateanimal cell, a mammal cell, or a human cell, a pig cell, a cow cell, asheep cell, a horse cell, a cat cell, a dog cell, a chicken cell, aturkey cell, a mouse cell, a rat cell, a hamster cell, a C. eleganscell, or a yeast cell. In certain embodiments, the detection may beperformed by measuring the level of Smek mRNA, the level of Smekprotein, or the level of a Smek-related activity.

In one aspect of the present invention, the above methods may be used toidentify a compound that inhibits the activity or expression of the Smekprotein by comparing the activity or expression of the Smek proteinafter contacting and the activity or expression of the Smek protein inthe absence of the compound to determine whether the compound inhibitsthe activity or expression of the Smek protein. Such aspect includes allembodiments of the above methods.

The present invention also includes methods of identifying a compoundthat increases the lifespan of an animal by enhancing phosphorylation ofa Smek protein. The method includes

-   -   contacting an isolated cell that expresses a Smek protein with a        compound;    -   detecting the phosphorylation level of the Smek protein; and    -   comparing the phosphorylation level of the Smek protein after        contacting and the phosphorylation level of Smek1 in the absence        of the compound to determine whether the compound enhances        phosphorylation of the Smek protein.

The above method includes all the above mentioned embodiments.

The present invention further includes methods of identifying a compoundthat inhibits phosphorylation of a Smek protein. The method includes

-   -   contacting an isolated cell that expresses the Smek protein with        a compound;    -   detecting the phosphorylation level of the Smek protein; and    -   comparing the phosphorylation level of the Smek1 protein after        contacting and the phosphorylation level of the Smek protein in        the absence of the compound to determine whether the compound        inhibits phosphorylation of the Smek protein.

The present invention further includes methods of identifying a compoundthat bind to a Smek protein. The method includes

-   -   contacting a Smek protein with a compound; and    -   measuring binding between the compound and the Smek protein.

The above aspects relating to methods of increasing the life span of ananimal and identifying compounds that increase the life span of ananimal may also be used to enhance the stress tolerance of an animal andidentify compounds that enhance the stress tolerance of an animal in allof the above embodiments and variations.

Another aspect of the present invention includes methods of inhibitingthe activity of Smek in a cell. The method includes

-   -   contacting a cell with an antisense or siRNA molecule.

In certain embodiments, the antisense molecule comprises apolynucleotide strand substantially complementary to a region of a Smekgene. In preferred embodiments, the antisense molecule is at least about75% identical to, at least about 80% identical to, at least about 85%identical to, at least about 90% identical to, at least about 95%identical to, at least about 97% identical to, or is identical to aregion of SEQ ID NO: 7, 8, 9, 10, or 11. In yet other embodiments, theregion is at least about 15 nucleotides long, at least about 20nucleotides long, at least about 25 nucleotides long, at least about 30nucleotides long, at least about 40 nucleotides long, at least about 50nucleotides long, or at least about 75 nucleotides long. In certainembodiments, the siRNA molecule comprises a first poly nucleotide strandthat is at least about 80% identical to, at least about 90% identicalto, at least about 95% identical to, or identical to a region of SEQ IDNO: 7, 8, 9, 10, or II and a second polynucleotide strand that is atleast about 80% identical to, at least about 90% identical to, at leastabout 95% identical to, or identical to a nucleotide sequencecomplementary to the region of SEQ ID NO: 7, 8, 9, 10, or 11,respectively. In various embodiments, the region is at least about 18,at least about 19, at least about 20, at least about 21, at least about22, or at least about 23 nucleotides long.

One aspect of the present invention includes stress-resistant non-humananimals comprising a transcriptional regulatory sequence active in theanimal operably linked to a recombinant nucleic acid encoding a Smekprotein. In a preferred embodiment, the Smek protein is Smek1. Morepreferably, the Smek1 protein is at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, or at least about 99% identical to the sequenceshown in SEQ ID NO: 1. In another embodiment, the Smek protein is Smek2.Preferably, the Smek2 protein is at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, or at least about 99% identical to the sequenceshown in SEQ ID NO: 2. In certain embodiments, the animal is avertebrate animal, a mammal, or a pig, a cow, a sheep, a horse, a cat, adog, a chicken, or a turkey. In various embodiments, the transcriptionalregulatory element may be heterologous to a Smek protein encodingrecombinant nucleic acid, and such element may promote constitutiveexpression, inducible expression or developmentally regulatedexpression.

Yet another aspect of the claimed invention includes stress-resistant,isolated animal cells comprising a transcriptional regulatory sequenceactive in the animal cell operably linked to a recombinant nucleic acidencoding a Smek protein. The stress resistant cell includes all of theabove variations and embodiments and includes human cells as well.

Another aspect of the present invention covers isolated stress inducedanimal cells comprising a Smek gene wherein Smek activity or expressionis repressed. In various embodiments, the animal cell may be avertebrate animal, a mammal, or a pig, a cow, a sheep, a horse, a cat, adog, a chicken, or a turkey cell. In certain embodiments, the Smek geneis the Smek1 gene and more preferred the Smek1 activity is specificallyrepressed. In certain other embodiments, the Smek gene is the Smek2 geneand more preferred the Smek2 activity is specifically repressed. Inpreferred embodiments, Smek activity is repressed at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, or at least about 75%.

Another aspect of the present invention covers isolated non-stressinduced animal cells comprising a Smek gene wherein Smek activity orexpression is elevated. In various embodiments, the animal cell may be avertebrate animal, a mammal, or a human, a pig, a cow, a sheep, a horse,a cat, a dog, a chicken, or a turkey cell. In certain embodiments, theSmek gene is the Smek1 gene and more preferred the Smek1 activity isspecifically elevated. In certain other embodiments, the Smek gene isthe Smek2 gene and more preferred the Smek2 activity is specificallyelevated. In preferred embodiments, Smek activity is elevated at leastabout 10%, at least about 20%, at least about 30%, at least about 50%,at least about 75%, at least about 100%, at least about 150%, at leastabout 250%, or at least about 500% above the normal level of activity.

In one embodiment, the present invention includes a Smek protein or anucleic acid molecule encoding a Smek protein. In a preferredembodiment, the Smek protein has the amino acid sequence set forth inSEQ ID NO: 1, 2, 3, 4, 5 or 6 or a conservative variant of the sequenceshown in SEQ ID NO: 1, 2, 3, 4, 5 or 6. In another preferred embodiment,the nucleic acid molecule has the sequence shown in SEQ ID NO: 7, 8, 9,10, or 11 or homologous sequence to the sequence shown in SEQ ID NO: 7,8, 9, 10, or 110. In certain embodiments, Smek protein is at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 97%, at least about 98%, or at least about 99%identical to the sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, or 6. Inother embodiments, Smek protein is encoded by a nucleotide sequence thathybridizes to SEQ ID NO: 7, 8, 9, 10, or 11 under very high stringencyhybridization, under high stringency hybridization, under moderatestringency hybridization or under low stringency hybridization. In stillother embodiments, Smek protein-encoding nucleic acid is at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 97%, at least about 98%, or at least about 99%identical to the sequence shown in SEQ ID NO: 7, 8, 9, 10, or 11.

In another embodiment, the present invention includes the above nucleicacids molecules operably linked to a promoter. In certain embodiments,the promoter may be a constitutive promoter, an inducible promoter, orregulated promoter such as a developmentally regulated, spatiallyregulated or temporally regulated promoter. In other embodiments, thepromoter is functional in animals, in vertebrates, or in mammals.Another embodiment of the present invention includes any of the abovenucleic acids in a vector or other genetic construct such as a viralgenome.

In still another embodiment, the present invention includes transgenicnon-human animals expressing a Smek protein as exemplified above orcomprising any of the above nucleic acids, vectors or other constructs.In certain embodiments, the expression of Smek protein may be limited toparticular developmental times, or particular tissues, such as duringadulthood, or in the white adipose tissue (WAT). In C. elegans, theintestine is an essential site of activity of DAF-16 (the FOXO homolog)and the nervous system is crucial site of activity of DAF-2. In mammals,recent experiments show that knockout of the insulin receptor in fattissue increases longevity and stress resistance. This tissue sharesmany similarities with the intestines of worms, where fat is stored inthis animal. In certain embodiments, the animals may be pigs, cows,sheep, horses, cats, dogs, chickens, or turkeys.

In one embodiment, the present invention is drawn to a method ofmodulating at least one trait in an animal which includes altering thelevel or the activity of Smek protein in an animal. In a preferredembodiment, the trait is longevity or stress resistance. In a preferredembodiment, Smek protein has the amino acid sequence set forth in SEQ IDNO. 1, 2, 3, 4, 5, or 6 or a conservative variant of the sequence shownin SEQ ID NO: 1, 2, 3, 4, 5, or 6. In certain embodiments, Smek proteinis at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 97%, at least about 50%, or at leastabout 99% identical to the sequence shown in SEQ ID NO: 1, 2, 3, 4, 5,or 6. In other embodiments, Smek protein is encoded by a nucleotidesequence that hybridizes to SEQ ID NO: 7, 8, 9, 10, or 11 under veryhigh stringency hybridization, under high stringency hybridization,under moderate stringency hybridization or under low stringencyhybridization.

In one embodiment, the level of Smek protein is altered by producing ananimal having an expression vector having a gene encoding Smek protein.Such animals shall preferably display either the trait of increasedlongevity or enhanced stress resistance. In a preferred embodiment, thegene encoding Smek protein has a nucleotide sequence that encodes theamino acid sequence set forth in SEQ ID NO. 1, 2, 3, 4, 5, or 6 or aconservative variant of the sequence shown in SEQ ID NO: 1, 2, 3, 4, 5,or 6. In another preferred embodiment, the gene encoding Smek proteinhas the nucleotide sequence set forth in SEQ ID NO. 7, 8, 9, 10, or 11.

In one embodiment, the present invention is drawn to a method ofmodulating a Smek-related trait in an animal. The method includes

-   -   transforming a animal cell with an expression vector including a        gene that encodes a Smek protein; and    -   culturing the animal cell into a animal under conditions that        allow the expression of the Smek protein thereby modulating a        Smek-related trait.

In a preferred embodiment, Smek protein is overexpressed in the animal.In a preferred embodiment, the Smek protein is encoded by a geneincluding the nucleotide sequence shown in SEQ ID NO: 7, 8, 9, 10, or11. In another preferred embodiment, Smek protein is encoded by a geneincluding the nucleotide sequence shown in SEQ ID NO: 7, 8, 9, 10, or11. In one preferred embodiment, the expression vector includes aconstitutive promoter. In an alternate preferred embodiment, theexpression vector includes an inducible promoter. In yet anotherembodiment, the expression vector includes a developmentally regulatedpromoter. Each of the foregoing promoters is operably linked to a Smekgene. In certain embodiments, the promoter may be heterologousincluding, without limitation, promoters from the same organism but adifferent gene and promoters from different organisms. In certainembodiments, the transgenic overexpression or modified expression isachieved by operably linking a heterologous promoter to an endogenousSmek protein gene.

In another aspect, the above described nucleic acids and vectors areoverexpressed in a cell. Preferably, the animal or animal cell is a pig,cow, sheep, horse, cat, dog, chicken, or turkey or a cell derived fromthe foregoing. In some preferred embodiments of methods not involving awhole transgenic animal, the animal or animal cell is a human or a humancell.

In a preferred embodiment, the Smek-related trait is a trait selectedfrom the group including: longevity, stress resistance, affinity forFOXO, transcription of stress related genes, and phosphorylation of theSmek protein. In a more preferred embodiment, the Smek-related trait islongevity, and the longevity is increased.

In one embodiment, the present invention is drawn to a method ofmodulating a Smek-related trait in an animal which includes contactingan animal cell, or animal, with an inhibitor or activator of a Smek genesuch that expression of the Smek gene is reduced or increased,respectively, compared to an animal not contacted with the inhibitor oractivator. Preferably, a Smek gene includes the nucleotide sequenceshown in SEQ ID NO: 7, 8, 9, 10, or 11. In another preferred embodiment,a Smek gene includes the nucleotide sequence shown in 7, 8, 9, 10, or11.

In a preferred embodiment, the inhibitor includes an expression vectorexpressing a protein, an antisense nucleic acid molecule or an siRNAthat inhibits expression of a Smek gene. In yet another preferredembodiment, the inhibitor is an siRNA molecule or an antisense nucleicacid molecule directed to a Smek gene, the p38γ MAP kinase gene, or thep38δ MAP kinase gene.

In a preferred embodiment, the Smek-related trait is a trait selectedfrom the group including longevity, stress resistance, affinity forFOXO, transcription of stress related genes, and phosphorylation of theSmek protein. In a more preferred embodiment, the Smek-related trait islongevity, and said longevity is increased. In certain embodiments, thelongevity is increased at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 75%,at least about 100%, at least about 150%, at least about 2000%, at leastabout 300%, or at least about 500%.

In one aspect, the present invention is drawn to a transgenic animalhaving at least one modulated Smek-related trait as compared to anon-transgenic animal, wherein the transgenic animal includes arecombinant expression vector that expresses a nucleic acid encoding aSmek gene. In a preferred embodiment, a Smek gene is overexpressed. In apreferred embodiment, a Smek gene includes the nucleotide sequence shownin SEQ ID NO: 7, 8, 9, 10 or 11. In a preferred embodiment, theexpression vector includes a constitutive promoter. In an alternatepreferred embodiment, the expression vector includes an induciblepromoter. In another preferred embodiment, the expression vectorincludes a developmentally regulated promoter. In each of the foregoing,the promoter is operably linked to a Smek gene.

In a preferred embodiment, the Smek-related trait in the transgenicanimal is a trait selected from the group including: longevity, stressresistance, affinity for FOXO, transcription of stress related genes,and phosphorylation of the Smek protein. In a more preferred embodiment,the Smek-related trait is longevity, and said longevity is increased.

In another aspect, the present invention includes methods of generatingrecombinant nucleic acid molecules encoding a Smek protein as well asthe recombinant nucleic acid molecules produced from such methods. Themethod includes providing genetic material from an animal and isolatingfrom the nuclear material the nucleic acid molecule encoding a Smekprotein. In various embodiments, the genetic material may be genomicDNA, RNA, cDNA generated from an animal. In certain embodiments, thegenetic material is encompassed in a library, which in certainembodiments may be an expression library. In certain embodiments, theanimal may be selected from the group including human, pig, cow, sheep,horse, cat, dog, chicken, or turkey. The nucleic acid molecule may beisolated by any method available to one of ordinary skill in the art. Incertain embodiments, the nucleic acid molecule is isolated byhybridization to a Smek encoding polynucleotide or fragment thereof.Examples of such isolation include hybridization to amplify the nucleicacid molecule, hybridization to identify the nucleic acid molecule in alibrary, and hybridization to directly purify the nucleic acid molecule.In another embodiment, the isolation is performed by screening anexpression library with an antibody to a Smek protein including withoutlimitation the Smek proteins disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A) Sequence alignment of Smek orthologs from human, Drosophila,C. elegans and S. cerevisiae. B) Localization of Smek1 and Smek2 in thehuman genome. C) Domain structure of human Smek1

FIG. 2: Localization of Smek1 isoforms in 293T cells. A) Nuclearlocalization of GFP-tagged Smek1. B) Immunofluorescence staining ofendogenous Smek1. C) Blocking of nuclear staining of Smek1 by antigen.D) Cytoplasmic localization of Smek1-S1-GFP. E) Nuclear translocation ofSmek1-S1-GFP after UV stimulation (180 J/m², 6 hrs). F) Control GFPlocalization after same UV treatment as in E).

FIG. 3: A) Dose-dependent phosphorylation of Smek1 upon osmotic stress.293T cells were stimulated with 0.3M and 0.6M sorbitol, respectively,lysed at different time points as indicated, followed by western blotanalysis using Smek1 antibodies. B) Dose-dependent phosphorylation ofSmek1 after UV treatment. HeLa cells were stimulated with different UVdosages and lysed after incubating for 1 hr at 37 degrees. C) Sustainedphosphorylation of Smek1 in response to UV stress. 293T cells weretreated with UV (180 J/m²), and cell lysates were collected every hourafterwards for 5 hrs followed by western blot analysis using Smek1antibodies. D) The phosphorylation of Smek1 induced by stress wasabolished by treating anti-Smek1 IPs with potato acid phosphatase (PAP).

FIG. 4: A) Lack of phosphorylation of GST-Smek1 by JNK MAPK in vitro.GST-cJUN was used a positive control for JNK activity. B)phosphorylation of GST-Smek1 by p38 MAPKs in vitro. Flag-tagged p38 MAPKisoforms were transfected into 293T cells, activated by stimulatingcells with UV (120 J/m²), and immunoprecipitated using anti-Flagantibodies for in vitro kinase assay. GST-ATF2 was used as a positivecontrol for p38 MAPK activity. The top panel showed the protein levelsof different p38 MAPK isoforms in the lysates. The lower panel showedthe differential phosphorylation of GST-Smek1 by p38 MAPKs. C)Identification of potential phosphorylation sites of Smek1. Top panelshowed the autoradiograph of p38 Kinase assay using GST-Smek andGST-Smek1-5A mutant as substrate, respectively. GST-ATF2 was thepositive control, and kinase inactive p38δ-KM and p38γ-AF were negativecontrols; middle panel showed the protein levels of p38δ and p38γ incell lysates; the bottom panel showed the predicted p38 MAP kinasephosphorylation sites in Smek1. D) Lack of phosphorylation of Smek1-5Amutant in response to stress in vivo. 293T cells transiently expressingFLAG-tagged Smek1-5A mutant were treated with various stress stimuli asindicated, and cell lysates were analyzed by western blotting incomparison to the wild type controls shown on the left.

FIG. 5: Interaction between Smek1 and FOXO proteins. A) Left panel: 293Tcells were transfected with FLAG-Smek1 in the absence or presence ofHA-FOXO3a were lysed for immunoprecipitation using anti-FLAG antibodies.The immunoprecipitates were resolved by SDS-PAGE and probed with anti-HAand anti-FLAG antibodies separately to show protein levels in the IPs(top) and lysates (bottom). Right panel: similar experiment wasperformed with Smek1, FOXO4 and FOXO4-TM mutant. The sample lanes werenumbered at the bottom for convenience. B) Left panel: 293T cells weretransfected with HA-FOXO3a in the absence or presence of FLAG-Smek1 orSmek1-5A mutant, followed by cell lysis, anti-FLAG immunoprecipitationand western blot analysis using anti-HA antibodies. IgG and α-tubulinwere used as controls for protein levels in IPs (top) and lysates(bottom), respectively. Right panel: the same blot was stripped andprobed with anti-FLAG antibodies to show Smek1 proteins levels in IPs(top) and lysates (bottom).

FIG. 6: Activation of FOXO3a-driven transcription by Smek1. A)Activation of a synthetic FOXO luciferase reporter by Smek1. HepG2hepatocytes were transfected with the indicated plasmids with asynthetic luciferase reporter containing three copies of FOXO bindingsites (pGL2-3xIRS) and a β-galactosidase reporter construct. Forty hourslater cell lysates were collected for luciferase assay and the data werenormalized to the value of β-galactosidase activity and presented as apercent of activity of vector control. B) Dosage-dependent activation ofFOXO reporter by Smek1. 293 cells were transfected with constitutivelyactive FOXO3a-TM mutant and various amount of Smek1 in the presence ofpGL2-3xIRS and a β-galactosidase reporter constructs. The data werenormalized to the value of β-galactosidase activity and presented asfold of the activity by expressing FOXO3a-TM alone. C) and D) Activationof native promoters of FOXO target gene by Smek1. Cells were transfectedas indicated together with a luciferase reporter driven by the nativepromoter of FOXO3a target genes, GADD45 and catalase, respectively. Thedata are shown as a percent of vector control calculated from duplicatedsamples.

FIG. 7: Working model. The figure shows two signaling pathways: (i) theinsulin/IGF-1-PI3K-AKT signaling pathway and (ii) the stress activatedpathway represented by the upstream kinase ASK1-downstream p38 MAPKcascade. The two pathways were shown to converge on a protein complexcontaining Smek1 and FOXO proteins in the nucleus. While AKTphosphorylation negatively regulates Smek1-FOXO interaction by excludingFOXO from the nucleus, stress signaling promotes the Smek1-FOXOinteraction via phosphorylation of both Smek1 and FOXO, which representsa balance that exists under physiological circumstances. As a result,the integrated response may be translated into changes in geneexpression that are important in stress resistance and life spanregulation.

FIGS. 8-13B: Additional Sequences. FIG. 8) shows the predictedDictyostelium (Dictyostelium discoideum) Smek 1 protein sequence (SEQ IDNO 26), FIGS. 9A-9B) show the Human Smek1 cDNA sequence (SEQ ID NO 27),FIGS. 10A-10B) show the Human Smek2 cDNA sequence (SEQ ID NO 28), FIGS.11A-11C) show the predicted Dictyostelium discoideum Smek1 cDNA sequence(SEQ ID NO 29), FIGS. 12A-12C) show the C. elegans Smek1 cDNA sequence(SEQ ID NO 30), FIGS. 13A-13B) show the S. cerevisiae Smek1 cDNAsequence (SEQ ID 31).

FIGS. 14A-14F. smk-1 is required for the increased longevity ofinsulin/IGF-1 signaling. In all cases, the solid black line depictsanimals grown on bacteria with an empty vector all of their life. Thesolid grey line depicts animals grown on bacteria producing smk-1 dsRNA.In cases where daf-16 RNAi was required, the cross-hatched line depictsanimals grown on bacteria expressing daf-16 RNAi. FIG. 14A) daf-2(e1370)long-lived mutant animals. FIG. 14B) N2, wild-type animals. FIG. 14C)isp-1(qm150) long-lived mutant animals. FIG. 14D) Long-lived cyc-1 RNAi(complex III) treated animals. FIG. 14E) daf-16(mu86) null mutantanimals. FIG. 14F) glp-1(e2141) long-lived mutant animals. Allstatistical data for life span analysis can be found in Table 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Embodiments of the invention are based, in part, upon the identificationof a Smek protein as the link between the stress response pathway andthe insulin/IGF signaling pathway. Thus, one embodiment of the inventionprovides isolated nucleic acids including nucleotide sequencescomprising or derived from Smek genes and/or encoding polypeptidescomprising or derived from Smek proteins. Smek sequences include thespecifically disclosed sequence, and splice variants, allelic variants,synonymous sequences, and homologous or orthologous variants thereof.Thus, for example, embodiments of the invention include genomic and cDNAsequences from a Smek gene.

Embodiments of the invention also include allelic variants andhomologous or orthologous sequences. For example, these variants areuseful in allele specific hybridization screening or PCR amplificationtechniques. Moreover, subsets of a Smek sequence, including both senseand antisense sequences, and both normal and mutant sequences, as wellas intronic, exonic and untranslated sequences, may be employed forthese techniques. Such sequences may comprise a small number ofconsecutive nucleotides from the sequence disclosed or otherwise enabledherein but preferably include at least 8-10, and more preferably 9-25,consecutive nucleotides from a Smek sequence. Various nucleic acidconstructs in which Smek sequences, either complete or subsets, areoperably joined to exogenous sequences to form cloning vectors,expression vectors, fusion vectors, transgenic constructs, and the likeare also contemplated.

Embodiments of the invention also include functional Smek polypeptides,and functional fragments thereof. As used herein, the term “functionalpolypeptide” refers to a polypeptide which possesses biological functionor activity which is identified through a defined functional assay andwhich is associated with a particular biologic, morphologic, orphenotypic alteration in the cell. The term “functional fragments ofSmek polypeptide”, refers to all fragments of Smek that retain Smekactivity, e.g., ability to confer a modulated Smek-related trait orparticular activities of the Smek protein such as the ability to bind toFOXO and the ability to enhance transcription by FOXO. Biologicallyfunctional fragments, for example, can vary in size from a polypeptidefragment as small as an epitope capable of binding an antibody moleculeto a large polypeptide capable of participating in the characteristicinduction or programming of phenotypic changes within a cell. A“Smek-related trait” refers to a trait that is mediated through a Smekprotein such as longevity, lifespan and response to stress.

Modifications of a Smek primary amino acid sequence may result in ananimal having reduced or abolished, or conversely an enhanced, Smekactivity. Such modifications may be deliberate, as by site-directedmutagenesis, or may be spontaneous. All of the polypeptides produced bythese modifications are included herein as long as the biologicalactivity of Smek is present. Further, deletion of one or more aminoacids can also result in a modification of the structure of theresultant molecule without significantly altering its activity. This canlead to the development of a smaller active molecule which could havebroader utility. For example, it may be possible to remove amino orcarboxy terminal amino acids without altering Smek activity.

Smek polypeptides include amino acid sequences substantially the same asthe sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, or 6. The term“substantially the same” refers to amino acid sequences that providenearly the same amino acid sequence, or retain the activity of Smek asdescribed herein. In preferred embodiments, the Smek protein is at leastabout 50%, at least about 60%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 97%, at least about 98%, or at least about 99%identical to the sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, or 6.Identity may be determined using any of the methods described hereinwhich align the polypeptides or fragments being compared and determinesthe extent of amino acid identity or similarity between them, e.g., byusing the publicly available program BLASTP. It will be appreciated thatamino acid “identity” is a comparison of amino acids that are identicalbetween two or more sequences being compared, which is different thanhomology which includes comparison of amino acids that are identical orare conserved variations. Identity may also be used in the context ofpolynucleotides, in which case one of skill in the art could use apublicly available program such as BLASTN. The Smek polypeptides of theinvention include conservative variations of the polypeptide sequence.

The term “conservative variation” as used herein denotes the replacementof an amino acid residue by another, biologically similar residue.Examples of conservative variations include the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another hydrophobic residue, or the substitution of one polarresidue for another polar residue, such as the substitution of argininefor lysine, glutamic for aspartic acids, or glutamine for asparagine,and the like. The term “conservative variation” also includes the use ofa substituted amino acid in place of an unsubstituted parent amino acidprovided that antibodies raised to the substituted polypeptide alsoimmunoreact with the unsubstituted polypeptide.

FIG. 1A shows the amino acid sequence alignment of several Smekproteins. The sequence alignment shows which regions of the protein aremore conserved than the others. In addition, one of skill in the art mayperform additional sequence alignments using other known methods. Suchsequence alignments provide a good indication of the degree of variationof amino acid residues at any given position that may be tolerated. Oneof skill in the art would understand that highly conserved regions maybe less able to tolerate significant variation and retain functionalactivity while less conserved regions may be able to tolerate variationand retain functional activity. Also, one of skill in the art willappreciate that where corresponding residues vary between the sequences,such variation gives an indication of the nature of changes that arelikely to be tolerated without disturbing the function of the protein.

Smek proteins can be analyzed by standard SDS-PAGE and/orimmunoprecipitation analysis and/or Western blot analysis, for example.Embodiments of the invention also provide an isolated polynucleotidesequence encoding a polypeptide having the amino acid sequence of SEQ IDNO: 7, 8, 9, 10, or 11 as well as nucleotide sequence encoding any ofthe above described Smek proteins. The term “isolated” as used hereinincludes polynucleotides or polypeptides, as applicable, substantiallyfree of other nucleic acids, proteins, lipids, carbohydrates or othermaterials with which they are naturally associated. Polynucleotidesequences of the invention include DNA, cDNA and RNA sequences whichencode Smek. It is understood that polynucleotides encoding all orvarying portions of Smek are included herein, as long as they encode apolypeptide with Smek activity. Such polynucleotides include naturallyoccurring, synthetic, and intentionally manipulated polynucleotides aswell as splice variants. For example, portions of the mRNA sequence maybe altered due to alternate RNA splicing patterns or the use ofalternate promoters for RNA transcription.

Moreover, Smek polynucleotides include polynucleotides havingalterations in the nucleic acid sequence that still encode a polypeptidehaving the ability to modulate a Smek-related trait such as longevity,lifespan and response to stress. Alterations in Smek nucleic acidinclude but are not limited to intragenic mutations (e.g., pointmutation, nonsense (stop), antisense, splice site and frameshift) andheterozygous or homozygous deletions. Detection of such alterations canbe done by standard methods known to those of skill in the art includingsequence analysis, Southern blot analysis, PCR based analyses (e.g.,multiplex PCR, sequence tagged sites (STSs)) and in situ hybridization.Embodiments of the invention also include anti-sense polynucleotidesequences.

The polynucleotides described herein include sequences that aredegenerate as a result of the genetic code. There are 20 naturallyoccurring amino acids, most of which are specified by more than onecodon. Therefore, all degenerate nucleotide sequences are included inthe invention as long as the amino acid sequence of Smek polypeptideencoded by such nucleotide sequences retains Smek activity A “functionalpolynucleotide” denotes a polynucleotide which encodes a functionalpolypeptide as described herein. In addition, embodiments of theinvention also include a polynucleotide encoding a polypeptide havingthe biological activity of an amino acid sequence of SEQ ID NO: 1, 2, 3,4, 5, or 6 and having at least one epitope for an antibodyimmunoreactive with Smek polypeptide.

As used herein, the terms “polynucleotides” and “nucleic acid sequences”refer to DNA, RNA and cDNA sequences and include all analogs andbackbone substitutes such as PNA that one of skill in the art wouldrecognize as capable of substituting for naturally occurring nucleotidesand backbones thereof.

Polynucleotides encoding Smek include the nucleotide sequence of SEQ IDNOS: 7, 8, 9, 10, and 11. cDNA sequences are shown in SEQ ID NO: 7, 8,9, 10, and 11. Nucleic acid sequences complementary to SEQ ID NOS: 7, 8,9, 10, and 11 are also encompassed within the present invention. Acomplementary sequence may include an antisense nucleotide. When thesequence is RNA, the deoxyribonucleotides A, G, C, and T of SEQ ID NOS:7, 8, 9, 10, or 11 are replaced by ribonucleotides A, G, C, and U,respectively. Also included in the invention are fragments (“probes”) ofthe above-described nucleic acid sequences that are at least 15 bases inlength, or preferably at least 16 bases in length, or preferably atleast 18 bases in length, or preferably at least 20 bases in length,which is sufficient to permit the probe to selectively hybridize to DNAthat encodes the protein of SEQ ID NO: 7, 8, 9, 10, or 11.

“Antisense” nucleic acids are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule(Weintraub, Scientific American 262 40, 1990). In the cell, theantisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule. This interferes with the translation of themRNA since the cell will not translate a mRNA that is double-stranded.Antisense oligomers of at least about 15 nucleotides are preferred, ofat least about 20 nucleotides are preferred, of at least about 25nucleotides are preferred, of at least about 30 nucleotides arepreferred, of at least about 35 nucleotides are preferred, of at leastabout 40 nucleotides are preferred, or of at least about 50 nucleotidesare preferred, since they are easily synthesized and are less likely tocause non-specific interference with translation than larger molecules.The use of antisense methods to inhibit the in vitro translation ofgenes is well known in the art (Marcus-Sakura Anal. Biochem. 172: 289,1998). In the present case, animals transformed with constructscontaining antisense fragments of the Smek gene would display amodulated Smek-related phenotype such as altered longevity.

Short double-stranded RNAs (dsRNAs; typically <30 nt) can be used tosilence the expression of target genes in animals and animal cells. Uponintroduction, the long dsRNAs enter the RNA interference (RNAi) pathwaywhich involves the production of shorter (20-25 nucleotide) smallinterfering RNAs (siRNAs) and assembly of the siRNAs into RNA-inducedsilencing complexes (RISCs). The siRNA strands are then unwound to formactivated RISCs, which cleave the target RNA. Double stranded RNA hasbeen shown to be extremely effective in silencing a target RNA.Introduction of double stranded RNA corresponding to the Smek gene wouldbe expected to modify the Smek-related traits discussed hereinincluding, but not limited to, longevity and stress tolerance.

“Hybridization” refers to the process by which a nucleic acid strandjoins with a complementary strand through base pairing. Hybridizationreactions can be sensitive and selective so that a particular sequenceof interest can be identified even in samples in which it is present atlow concentrations. Suitably stringent conditions can be defined by, forexample, the concentrations of salt or formamide in the prehybridizationand hybridization solutions, or by the hybridization temperature, andare well known in the art. In particular, stringency can be increased byreducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS,and 200 ng/ml sheared and denatured salmon sperm DNA. Hybridizationcould occur under medium stringency conditions as described above, butin 35% formamide at a reduced temperature of 35° C. The temperaturerange corresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art. “Selectivehybridization” as used herein refers to hybridization under moderatelystringent or highly stringent physiological conditions (See, forexample, the techniques described in Maniatis et al., 1989 MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.,incorporated herein by reference), which distinguishes SMEK-relatedsequences from unrelated nucleotide sequences.

In another aspect of the invention, very high stringency hybridizationconditions can include at least one wash at 0.1×SSC, 0.1% SDS, at 60° C.for 15 minutes. High stringency hybridization conditions can include atleast one wash at 0.2×SSC, 0.1% SDS, at 60° C. for 15 minutes. Moderatestringency hybridization conditions can include at least one wash at0.5×SSC, 0.1% SDS, at 60° C. for 15 minutes. Low stringencyhybridization conditions can include at least one wash at 1.0×SSC, 0.1%SDS, at 60° C. for 15 minutes.

Another aspect of the invention is polypeptides or fragments thereofwhich have at least about 70%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or more than about 95% homologyto SEQ ID NO: 1, 2, 3, 4, 5, or 6, and sequences substantially identicalthereto, or a fragment comprising at least 5, 10, 15, 20, 25, 30, 35,40, 50, 75, 100, or 150 consecutive amino acids thereof. Homology may bedetermined using any of the methods described herein which align thepolypeptides or fragments being compared and determines the extent ofamino acid identity or similarity between them. It will be appreciatedthat “homology” includes polypeptides having conservative amino acidsubstitutions such as those described above. “Homolog” includes a generelated to a second gene by descent from a common ancestral DNAsequence. The term, homolog, may apply to the relationship between genesseparated by the event of speciation or to the relationship betweengenes separated by the event of genetic duplication. “Orthologs” aregenes in different species that evolved from a common ancestral gene byspeciation. Normally, orthologs retain the same function in the courseof evolution. Identification of orthologs is critical for reliableprediction of gene function in newly sequenced genomes.

The polypeptides or fragments having homology to SEQ ID NO: 1, 2, 3, 4,5, or 6, and sequences substantially identical thereto, or a fragmentcomprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or150 consecutive amino acids thereof may be obtained by isolating thenucleic acids encoding them using the techniques described herein.

Alternatively, the homologous polypeptides or fragments may be obtainedthrough biochemical enrichment or purification procedures. The sequenceof potentially homologous polypeptides or fragments may be determined byproteolytic digestion, gel electrophoresis and/or microsequencing. Thesequence of the prospective homologous polypeptide or fragment can becompared to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, or 6, andsequences substantially identical thereto, or a fragment comprising atleast about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof using any of the programs describedabove.

Also included in embodiments of the invention are nucleotide sequencesthat are greater than 70% homologous with SEQ ID NOS: 7, 8, 9, 10, or11, but still retain the ability to modulate a Smek-related trait suchas longevity, lifespan and stress tolerance. Other embodiments of theinvention include nucleotide sequences that are greater than 75%, 80%,85%, 90% or 95% homologous with SEQ ID NOS: 7, 8, 9, 10, or 11, butstill retain the ability to confer a modulated Smek-related trait whichincludes altered longevity, lifespan and stress tolerance.

Also included in embodiments of the invention are nucleotide sequencesthat are greater than 70% identical to SEQ ID NOS: 7, 8, 9, 10, or II,but still retain the ability to modulate a Smek-related trait such aslongevity. Other embodiments of the invention include nucleotidesequences that are greater than 75%, 80%, 85%, 90% or 95% identical toSEQ ID NOS: 7, 8, 9, 10, or 11, but still retain the ability to confer amodulated Smek-related trait which includes altered longevity.

Specifically disclosed herein is a cDNA sequence for Smek as well as twogenomic DNA sequences. DNA sequences of the invention can be obtained byseveral methods. For example, the DNA can be isolated usinghybridization or computer-based techniques which are well known in theart. Such techniques include, but are not limited to: 1) hybridizationof genomic or cDNA libraries with probes to detect homologous nucleotidesequences; 2) antibody screening of expression libraries to detectcloned DNA fragments with shared structural features; 3) polymerasechain reaction (PCR) on genomic DNA or cDNA using primers capable ofannealing to the DNA sequence of interest; 4) computer searches ofsequence databases for similar sequences; and 5) differential screeningof a subtracted DNA library.

Screening procedures which rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate probe is available. Oligonucleotide probes, which correspondto a par of the Smek sequence encoding the protein in question, can besynthesized chemically. This requires that short, oligopeptide stretchesof the amino acid sequence must be known. The DNA sequence encoding theprotein can be deduced from the genetic code, however, the degeneracy ofthe code must be taken into account. It is possible to perform a mixedaddition reaction when the sequence is degenerate. This includes aheterogeneous mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. Hybridization isparticularly useful in the detection of cDNA clones derived from sourceswhere an extremely low amount of mRNA sequences relating to thepolypeptide of interest are present. In other words, by using stringenthybridization conditions directed to avoid non-specific binding, it ispossible, for example, to allow the autoradiographic visualization of aspecific cDNA clone by the hybridization of the target DNA to thatsingle probe in the mixture which is its complete complement (Wallace,et al., Nucl. Acid Res. 9, 879, 1981). Alternatively, a subtractivelibrary is useful for elimination of non-specific cDNA clones.

Among the standard procedures for isolating cDNA sequences of interestis the formation of plasmid- or phage-carrying cDNA libraries which arederived from reverse transcription of mRNA which is abundant in donorcells that have a high level of genetic expression. When used incombination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al. Nucl. Acid Res, 11, 2325, 1983).

A cDNA expression library, such as lambda gt11, can be screenedindirectly for Smek peptides using antibodies specific for Smek. Suchantibodies can be either polyclonally or monoclonally derived and usedto detect expression product indicative of the presence of Smek cDNA.

Another embodiment of the invention relates to animals that have atleast one modulated Smek-related trait. Such modulated traits includeamong others an altered longevity and an altered stress tolerance,“Longevity” refers to the life span of the animal. Thus, longevityrefers to the number of years in the life span of an animal. “Stresstolerance” refers to an animal's ability to tolerate exposure to variousinternal and external environmental challenges such as exposure to UVlight, exposure to high osmolarity, exposure to infection, exposure tooxidative damage, exposure to metal compounds, and exposure to certaintoxins. Those of skill in the art will recognize that an increase in thelifespan of an animal can readily be measured by various assays known inthe art. The field of gerontology is one such example of a relevant art.By way of example, longevity may be assessed by various markers such asnumber of generations to senescence in non-immortalized somatic cells,graying hair, wrinkling, and other such alterations physiologicalmarkers associated with aging. Those of skill in the art will alsorecognize that alterations in an animals ability to tolerate stress,i.e., its response to stress, may be assessed by various assays,including by way of example, by assessing changes in expression oractivity of molecules involved in the stress response by measuringexpression of stress response genes, protein levels of specific stressresponse proteins, or activity levels of specific stress responseproteins.

Animals having a modified Smek-related trait include transgenic animalswith an altered longevity or an altered stress tolerance due totransformation with constructs using antisense or siRNA technology thataffect transcription or expression from a Smek gene. Such animalsexhibit an altered longevity (or life span) and an altered stresstolerance.

Accordingly, in another series of embodiments, the present inventionprovides methods of screening or identifying proteins, small moleculesor other compounds which are capable of inducing or inhibiting theactivity or expression of Smek genes and proteins. The assays may beperformed, by way of example, in vitro using transformed ornon-transformed cells, immortalized cell lines, or in vivo usingtransformed animal models enabled herein. An example of a preferredanimal model would be a transgenic mouse with one or both of theendogenous Smek genes replaced with the corresponding human Smek genes.In particular, the assays may detect, for example, the presence ofincreased or decreased activity or expression of Smek (from human orother animal) genes or proteins on the basis of increased or decreasedmRNA expression, increased or decreased levels of Smek protein products,or increased or decreased levels of expression of a marker gene (e.g.,beta-galactosidase, green fluorescent protein, alkaline phosphatase orluciferase) operably joined to an Smek 5′ regulatory region in arecombinant construct, increased or decreased phosphorylation of theSmek protein, increased or decreased affinity for FOXO proteins. Cellsknown to express a particular Smek, or transformed to express aparticular Smek, are incubated and one or more test compounds are addedto the medium under conditions in which Smek nucleic acid or protein isknow to be modulated. In addition, in higher organisms with at least twoSmek genes, such as humans, compounds that selectively induce or inhibitthe activity or expression of one Smek protein and not another may beidentified in such assays. Such assays could, for example, use pairs ofcell-lines, each only expressing one such Smek gene and comparing theeffect of the compound on each cell-line. After allowing a sufficientperiod of time (e.g., 0-72 hours) for the compound to induce or inhibitthe activity or expression of Smek, any change in levels of activity orexpression from an established baseline may be detected using any of thetechniques described above.

In another series of embodiments, the present invention provides methodsfor identifying proteins and other compounds which bind to, or otherwisedirectly interact with Smek protein. The proteins and compounds includeendogenous cellular components which interact with Smek in vivo andwhich, therefore, provide new targets for therapeutic or diagnosticproducts, as well as recombinant, synthetic and otherwise exogenouscompounds which may have Smek binding capacity and, therefore, arecandidates for modulating Smek-related traits. In addition, in higherorganisms with at least two Smek proteins, such as humans, compoundsthat selectively bind to one protein and not the other may be identifiedin such assays. Such assays could use parallel or sequential bindingassays against the two proteins. Thus, in one series of embodiments,High Throughput Screening-derived proteins, DNA chip arrays, celllysates or tissue homogenates may be screened for proteins or othercompounds which bind to one of the normal or mutant Smek genes.Alternatively, any of a variety of exogenous compounds, both naturallyoccurring and/or synthetic (e.g., libraries of small molecules orpeptides), may be screened for Smek binding capacity.

In each of these embodiments, an assay is conducted to detect bindingbetween Smek and another moiety. Smek in these assays may be anypolypeptide comprising or derived from a normal or mutant Smek protein,including functional domains or antigenic determinants of Smek. Bindingmay be detected by non-specific measures (e.g., transcriptionmodulation, altered chromatin structure, peptide production or changesin the expression of other downstream genes which can be monitored bydifferential display, 2D gel electrophoresis, differentialhybridization, or SAGE methods) or by direct measures such asimmunoprecipitation, the Biomolecular Interaction Assay (BIAcore) oralteration of protein gel electrophoresis. The preferred methods involvevariations on the following techniques: (1) direct extraction byaffinity chromatography; (2) co-isolation of Smek components and boundproteins or other compounds by immunoprecipitation; (3) BIAcoreanalysis; and (4) the yeast two-hybrid systems.

Embodiments of the invention also include methods of identifyingproteins, small molecules and other compounds capable of modulating theactivity of normal or mutant Smek. Using normal cells or animals, thetransformed cells and animal models of the present invention, or cellsobtained from subjects bearing normal or mutant Smek genes, the presentinvention provides methods of identifying such compounds on the basis oftheir ability to affect the expression of Smek, the activity of Smek,the activity of other Smek-regulated genes, the activity of proteins,such as FOXO, that interact with normal or mutant Smek proteins, theintracellular localization of Smek, changes in transcription activity,the presence or levels of membrane bound Smek, the level ofphosphorylation of Smek, or other biochemical, histological, orphysiological markers which distinguish cells bearing normal andmodulated Smek activity in animals.

In accordance with another aspect of the invention, the proteins of theinvention can be used as starting points for rational chemical design toprovide ligands or other types of small chemical molecules.Alternatively, small molecules or other compounds identified by theabove-described screening assays may serve as “lead compounds” in designof modulators of Smek-related traits in animals.

DNA sequences encoding Smek can be expressed in vitro by DNA transferinto a suitable host cell. “Host cells” are cells in which a vector canbe propagated and its DNA expressed. The term also includes any progenyor graft material, for example, of the subject host cell. It isunderstood that all progeny may not be identical to the parental cellsince there may be mutations that occur during replication. However,such progeny are included when the term “host cell” is used. Methods ofstable transfer, meaning that the foreign DNA is continuously maintainedin the host, are known in the art.

As part of the present invention, Smek polynucleotide sequences may beinserted into a recombinant expression vector. The terms “recombinantexpression vector” or “expression vector” refer to a plasmid, virus orother vehicle known in the art that has been manipulated by insertion orincorporation of a Smek genetic sequence. Such expression vectorscontain a promoter sequence which facilitates the efficienttranscription of the inserted Smek sequence. The expression vectortypically contains an origin of replication, a promoter, as well asspecific genes which allow phenotypic selection of the transformedcells.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing Smek coding sequence andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo recombination/genetic techniques.

A variety of host-expression vector systems may be utilized to express aSmek coding sequence. These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining a Smek coding sequence; yeast transformed with recombinantyeast expression vectors containing a Smek coding sequence; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing a Smek coding sequence; insect cell systems infectedwith recombinant virus expression vectors (e.g., baculovirus) containinga Smek coding sequence; or animal cell systems infected with recombinantvirus expression vectors (e.g., retroviruses, adenovirus, vacciniavirus) containing a Smek coding sequence, or transformed animal cellsystems engineered for stable expression.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation elements, including constitutiveand inducible promoters, transcription enhancer elements, transcriptionterminators, etc. may be used in the expression vector (see e.g., Bitteret al. Methods in Enzymology 153, 516-544, 1987). For example, whencloning in bacterial systems, inducible promoters such as pL ofbacteriophage 7, plac, ptrp, ptac (ptrp-lac hybrid promoter) and thelike may be used. When cloning in mammalian cell systems, promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the retrovirus long terminalrepeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter)may be used. Promoters produced by recombinant DNA or synthetictechniques may also be used to provide for transcription of the insertedSmek coding sequence.

Isolation and purification of recombinantly expressed polypeptide, orfragments thereof, may be carried out by conventional means includingpreparative chromatography and immunological separations involvingmonoclonal or polyclonal antibodies. In addition, the recombinantlyexpressed polypeptide, of fragments thereof, may include an affinitytag, such as a FLAG-tag, a his-tag, a GST or MBP fusion. Such affinitytags may be preferable when the polypeptide is to be used inidentification of compounds that bind to Smek or modulate the activityof Smek owing to the ease of manipulation.

In another embodiment of the invention provide a method for producing agenetically modified non-human animal having at least one modulatedSmek-related trait such as having an altered longevity as compared to ananimal which has not been genetically modified. One of skill in the artwill recognize that a Smek-related traits such as longevity and stresstolerance may vary from individual to individual, so the average overseveral individuals in a population needs to be determined whencomparing such traits. The method includes the steps of contacting ananimal cell with at least one vector containing at least one nucleicacid sequence encoding a Smek gene or a mutant, homolog or fragmentthereof, wherein the nucleic acid sequence is operably associated with apromoter or a transcriptional regulatory element, to obtain atransformed animal cell; producing a transgenic animal from thetransformed animal cell; and thereafter selecting an animal exhibiting amodulated Smek-related trait such as an altered longevity. One of skillin the art will appreciate that the present invention also includestransgenic modulation of endogenous Smek gene expression by introducinga heterologous promoter or transcriptional regulatory element into thegenome of an animal such that the promoter or element is operably linkedto the Smek gene.

Transgenic animals that result in at least one modulated Smek-relatedtrait such as an altered longevity may be obtained by reduced expressionof the Smek gene. Thus, one embodiment of the invention includes animalstransformed with antisense polynucleotides complementary to a Smek geneor fragments thereof wherein production of the antisense polynucleotidesresults in reduced expression of the Smek gene. In an alternateembodiment, reduced expression of Smek may also be achieved by methodssuch as expression of siRNAs targeting a Smek gene by operativelylinking an siRNA gene to a promoter. In an alternate embodiment,transgenic animals overexpressing a Smek gene are described. Suchanimals might be expected to display a modulated Smek-related trait suchas an altered longevity, lifespan, or stress tolerance.

The term “genetic modification” as used herein refers to theintroduction of one or more heterologous nucleic acid sequences, e.g., aSmek sequence or a Smek mutant encoding sequence, into one or moreanimal cells, which can generate whole, adult animal by nucleartransplantation or pronuclear injection into an embryo or oocyte andimplantation of such embryo or oocyte into the uterus of a host animal.The term “genetically modified” as used herein refers to an animal whichhas been generated through the aforementioned process. Geneticallymodified animals of the invention are capable of interbreeding withother animals of the same species so that the foreign gene, carried inthe germ line, can be inserted into or bred into agriculturally usefulanimal varieties. The term “animal cell” as used herein refers toimmortalized cell lines, embryonic stem cells, and non-immortalized celllines. Accordingly, an embryo comprising multiple animal cells capableof developing to term into an adult animal, is included in thedefinition of “animal cell”.

As used herein, the term “animal” refers to either a whole animal, ananimal organ, an animal cell, or a group of animal cells, such as ananimal tissue, for example, depending upon the context. Animals includedin the invention are any animals amenable to transformation techniques,including vertebrate and non-vertebrate animals and mammals. Examples ofmammals include, but are not limited to, pigs, cows, sheep, horses,cats, dogs, chickens, or turkeys.

The term “exogenous nucleic acid sequence” as used herein refers to anucleic acid foreign to the recipient animal host or, native to the hostif the native nucleic acid is substantially modified from its originalform. For example, the term includes a nucleic acid originating in thehost species, where such sequence is operably linked to a promoter thatdiffers from the natural or wild-type promoter. In one embodiment, atleast one nucleic acid sequence encoding Smek or a variant thereof isoperably linked with a promoter. It may be desirable to introduce morethan one copy of a Smek polynucleotide into an animal for enhancedexpression. For example, multiple copies of the gene would have theeffect of increasing production of the Smek gene product in the animal.

Genetically modified animals of the present invention are produced bycontacting an animal cell with a vector including at least one nucleicacid sequence encoding a Smek or a variant thereof. To be effective onceintroduced into animal cells, a Smek nucleic acid sequence is operablyassociated with a promoter which is effective in the animal cell tocause transcription of Smek. Additionally, a polyadenylation sequence ortranscription control sequence, also recognized in animal cells may alsobe employed. It is preferred that the vector harboring the nucleic acidsequence to be inserted also contain one or more selectable marker genesso that the transformed cells can be selected from non-transformed cellsin culture, as described herein.

The term “operably linked” refers to functional linkage between apromoter sequence and a nucleic acid sequence regulated by the promoter.The operably linked promoter controls the expression of the nucleic acidsequence.

The expression of structural genes may be driven by a number ofpromoters. Although the endogenous, or native promoter of a structuralgene of interest may be utilized for transcriptional regulation of thegene, preferably, the promoter is a foreign regulatory sequence. Formammalian expression vectors, promoters capable of directing expressionof the nucleic acid preferentially in a particular cell type may be used(e.g., tissue-specific regulatory elements are used to express thenucleic acid). Tissue-specific regulatory elements are known in the art.Non-limiting examples of suitable tissue-specific promoters include thealbumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv.Immunol. 43: 235-275), in particular promoters of T cell receptors(Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins(Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983.Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilamentpromoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science230: 912-916), and mammary gland-specific promoters (e.g., milk wheypromoter; U.S. Pat. No. 4,873,316 and European Application PublicationNo. 264,166). Developmentally-regulated promoters are also encompassed,e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989.Genes Dev. 3: 537-546).

Promoters useful in the invention include both natural constitutive andinducible promoters as well as engineered promoters. To be most useful,an inducible promoter should 1) provide low expression in the absence ofthe inducer; 2) provide high expression in the presence of the inducer;3) use an induction scheme that does not interfere with the normalphysiology of the animal; and 4) have no effect on the expression ofother genes. Examples of inducible promoters useful in animals includethose induced by chemical means, such as the yeast metallothioneinpromoter which is activated by copper ions (Mett, et al. Proc. Natl.Acad. Sci., U.S.A. 90, 4567, 1993); and the GRE regulatory sequenceswhich are induced by glucocorticoids (Schena, et al. Proc. Natl. Acad.Sci., U.S.A. 88, 10421, 1991). Other promoters, both constitutive andinducible will be known to those of skill in the art.

The particular promoter selected should be capable of causing sufficientexpression to result in the production of an effective amount ofstructural gene product to modulate a Smek-related trait such aslongevity. The promoters used in the vector constructs of the presentinvention may be modified, if desired, to affect their controlcharacteristics.

Optionally, a selectable marker may be associated with the nucleic acidsequence to be inserted. As used herein, the term “marker” refers to agene encoding a trait or a phenotype which permits the selection of, orthe screening for, an animal or animal cell containing the marker.Preferably, the marker gene is an antibiotic resistance gene whereby theappropriate antibiotic can be used to select for transformed cells fromamong cells that are not transformed. Suitable markers will be known tothose of skill in the art.

Vector(s) employed in the present invention for transformation of aanimal cell include a nucleic acid sequence encoding Smek, operablylinked to a promoter. To commence a transformation process in accordancewith the present invention, it is first necessary to construct asuitable vector and properly introduce it into the animal cell. Detailsof the construction of vectors utilized herein are known to thoseskilled in the art of animal genetic engineering.

A transgenic animal of the present invention can be created byintroducing Smek protein-encoding nucleic acid into the male pronucleiof a fertilized oocyte (e.g., by microinjection, retroviral infection)and allowing the oocyte to develop in a pseudopregnant female fosteranimal. Sequences including SEQ ID NO: 7, 8, 9, 10, or 11 can beintroduced as a transgene into the genome of a non-human animal.Alternatively, a non-human homolog of the human Smek gene, such as amouse Smek1 gene, can be isolated based on hybridization to the humanSmek1 gene and used as a transgene. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably-linked to theSmek transgene to direct expression of Smek protein to particular cells.Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan, 1986. In:Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. Similar methods are used for production of othertransgenic animals. A transgenic founder animal can be identified basedupon the presence of the Smek transgene in its genome and/or expressionof Smek mRNA in tissues or cells of the animals. A transgenic founderanimal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene-encodingSmek protein can further be bred to other transgenic animals carryingother transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a Smek gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the Smek gene. The Smek gene can be a human gene(e.g., the DNA of SEQ ID NO: 7 or 8), but may also be a non-humanhomolog of a human Smek gene. For example, a mouse homolog of the humanSmek1 gene or Smek2 gene of SEQ ID NO: 7 or 8 can be used to construct ahomologous recombination vector suitable for altering an endogenous Smekgene in the mouse genome. In one embodiment, the vector is designed suchthat, upon homologous recombination, the endogenous Smek gene isfunctionally disrupted (i.e., no longer encodes a functional protein;also referred to as a “knock out” vector).

Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous Smek gene is mutated or otherwise alteredbut still encodes functional protein (e.g., the upstream regulatoryregion can be altered to thereby alter the expression of the endogenousSmek protein). In the homologous recombination vector, the alteredportion of the Smek gene is flanked at its 5′- and 3′-termini byadditional nucleic acid of the Smek gene to allow for homologousrecombination to occur between the exogenous Smek gene carried by thevector and an endogenous Smek gene in an embryonic stem cell. Theadditional flanking Smek nucleic acid is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′- and 3′-termini) areincluded in the vector. See, e.g., Thomas, et al., 1987. Cell 51: 503for a description of homologous recombination vectors. The vector isthen introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced Smek gene hashomologously-recombined with the endogenous Smek gene are selected. See,e.g., Li, et al., 1992. Cell 69: 915.

The selected cells are then injected into a blastocyst of an animal(e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987.In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously-recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously-recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley, 1991. Curr. Opin. Biotechnol. 2: 823-829; PCTInternational Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968;and WO 93/04169.

In another embodiment, transgenic non-humans animals can be producedthat contain selected systems that allow for regulated expression of thetransgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, See, e.g., Lakso, et al., 1992. Proc. Natl. Acad.Sci. USA 89: 6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, etal., 1991. Science 251:1351-1355. If a Cre/loxP recombinase system isused to regulate expression of the transgene, animals containingtransgenes encoding both the Cre recombinase and a selected protein arerequired. Such animals can be provided through the construction of“double” transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, et al., 1997.Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G₀ phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to morula orblastocyst and then transferred to pseudopregnant female foster animal.The offspring borne of this female foster animal will be a clone of theanimal from which the cell (e.g., the somatic cell) is isolated

As used herein, the term “contacting” refers to any means of introducingSmek into an animal cell, including chemical and physical means asdescribed above.

Transgenic animals exhibiting a modulated Smek-related trait such as anincreased life span or an enhanced stress tolerance as compared withnon-transgenic animals can be selected by observation. While life spanvaries from animal to animal the average life span can be observed byaveraging the life span of several examples of the animal. Stresstolerance can be measured by exposing an animal to various levels ofstress and measuring the response, and comparing to the stress responsein non-transgenic animals. The invention includes animal produced by themethod of the invention, as well as animal tissues and animal cells.

In yet another embodiment, the invention provides a method for producinga genetically modified animal cell such that an animal produced from thecell has a modulated Smek-related trait such as an increased life spancompared with a non-transgenic animal. The method includes contactingthe animal cell with an Smek nucleic acid sequence to obtain atransformed animal cell; transferring the nucleus of the transformedanimal cell into an oocyte; implanting the oocyte into the uterus of ananimal and allowing the transgenic animal to develop to term to obtain atransgenic animal having a modulated Smek-related trait such as anincreased life span.

It will be understood by those of skill in the art that numerous andvarious modifications can be made without departing from the spirit ofthe present invention. A more complete understanding can be obtained byreference to the following specific examples which are provided hereinfor purposes of illustration only and are not intended to limit thescope of the invention.

EXAMPLES

The following examples include the characterization of a novel proteinSmek1 (Suppressor of mek null), which belongs to a protein familyconserved among eukaryotic organisms from yeast to mammals. The examplesreveal that Smek1 is a nuclear target for a p38 MAP kinase-relatedstress response pathway in mammalian cells. To define the biologicalfunction of Smek1, RNAi in C. elegans was used to deplete the singleSmek1 homolog. This analysis indicated that worm Smek1 plays a role inregulating stress resistance and organismal longevity (See Example 6).Since Smek1 appears to function in parallel to the FOXO homolog Daf-16,the key regulator of stress resistance and longevity (Kenyon 2001) andSmek1 binds to FOXO in mammalian cells as demonstrated below, Smek1 andFOXO function as a complex. Example 4 demonstrated that Smek1 indeedinteracts with forkhead transcription factors (FOXOs) in mammaliancells.

The following examples further indicate that Smek1 is regulated byphosphorylation in response to stress, while FOXO proteins arewell-defined downstream targets for Akt kinase, which has been shown toinhibit transcriptional activity of FOXO by causing its cytoplasmicretention upon phosphorylation (Brunet, Bonni et al. 1999).Interestingly, Example 2 indicates that stress-induced phosphorylationof Smek1 and phosphorylation of FOXO via insulin-PI3K-Akt pathway playan opposing role in regulating Smek1-FOXO interaction, showing themolecular mechanism for the stress response pathway and insulin/IGF-1signaling pathway to crosstalk and counteract one another in relevantbiological processes. Furthermore, Example 5 shows that Smek1 is able toregulate gene expression in part by promoting FOXO-driven transcription.Thus, a protein complex containing Smek1 and FOXO arose during evolutionto serve as a nodal point to integrate signals from insulin/IGF-1pathway and stress response pathway, and perhaps other signalingpathways. Consequently the status of this complex in turn determines therelevant gene expression output that underlies physiological phenomenasuch as aging. Thus, the present invention focuses on the Smek proteinas a target for modulation of aging and other important physiologicalphenomena.

Finally, the examples include genetic analysis that indicates that lossof smk-1 specifically influences the aging related function of the DAF-2Insulin/IGF-1 signaling pathway. Localization analysis of DAF-16 placesSMK-1 downstream of DAF-16's phosphorylation-dependent relocation to thenucleus, transcriptional assays indicate that SMK-1 is required formaximal DAF-16/FOXO3a transcription, and physiological evidence suggeststhat DAF-16 and SMK-1 are capable of functional interaction in thenuclei of intestinal cells and neurons. Taken together, the examplesindicate that SMK-1 is a new component of the Insulin/IGF-1 signalinglongevity pathway, and the first that plays a role in longevity withoutaffecting other processes regulated by Insulin/IGF-1 signaling,presumably by modulating DAF-16 transcriptional specificity.

Example 1 Smek Belongs to a Conserved Novel Protein Family

Smek (suppressor of mek1 null) was identified from Dictyostelium in asecond site suppressor screen in a null strain background defective inthe MAP kinase DdMEK1. Loss of DdSmek partially suppressed thechemotaxis and developmental defects of Dictyostelium mekl null cells(unpublished data). Based on the results of Genbank database searches,the Smek orthologs comprise a novel gene family conserved in diverseeukaryotic organisms including yeast, fly, worm, plant and mammals (FIG.1A). There are two Smek genes in the human genome (Smek1 and Smek2),which are localized on the chromosome 14 and chromosome 2, respectively(FIG. 1B). In addition, an intron-less pseudogene was identified on theX chromosome, which does not have any matching EST clones.

Human Smek1 is composed of 820 amino acid residues. Similar to otherSmek homologs, the only region of Smek1 that shares significant homologyto any identified protein domain is the N-terminal region (approximatelyresidues 1-100) EVH1 domain, a domain known to bind proline-richsequences (Volkman, Prehoda et al. 2002) (FIG. 1C). The central regionof Smek1 (approximately residues 200-600) is highly hydrophobic andcontains a novel domain DUF625 (Domain of Unidentified Function), whichappears to be conserved only among Smek orthologs. The C-terminal regionof Smek1 is rich in charged residues and is more divergent among Smekhomologs. According to the diverse sources of EST clones in thedatabase, human Smek1, as well as Smek2, are widely expressed in varioustissues, including brain, liver, pancreas, kidney, testis, ovary andbreast. Furthermore, multiple Smek1 EST sequences were identified tocontain stop codons within the junction of central and C-terminaldomains, demonstrating that Smek1 transcripts are regulated byalternative splicing.

Example 2 SMEK1 is a Stress Response Protein

First, the localization of Smek1 in the cell was investigated bytransiently expressing a C-terminal GFP tagged full-length Smek1 incultured mammalian cells, which revealed that Smek1 is localized in thenucleus (FIG. 2A). This observation was confirmed by immunofluorescencestaining of the endogenous protein using an affinity-purified antiserumraised against Smek1 (FIG. 2B). The nuclear staining of Smek1 antibodywas effectively blocked by incubating the serum with correspondingantigen (FIG. 2C). Interestingly, the GFP fusion of a natural Smek1isoform with a truncated C-terminus (Smek1-S1) localized exclusively inthe cytoplasm (FIG. 2D), indicating the presence of potential NLS(nuclear localization signal) within the C-terminus.

The conditions under which cytoplasmic Smek1-S1 can translocate into thenucleus were identified. Various conditions were tested with culturedmammalian cell lines expressing a GFP-tagged Smek1-S1, including serumstarvation, growth factor stimulation, stress treatments and variationsof glucose concentrations in the medium. These experiments revealed thatwhen cells were exposed to a high dose of UV irradiation, Smek1-S1 wastranslocated into the nucleus (FIG. 2E), demonstrating that Smek1proteins are involved in the stress response pathway. Similar nucleartranslocation of Smek1-S1 was observed in cells treated with highosmolarity (data not shown).

Given that full length Smek1 contains multiple potential phosphorylationsites, especially a cluster of five Serine-Proline (SP) sites in theC-terminal region, Smek1 is regulated by phosphorylation, in particularby stress-activated MAP kinases. Western blot analysis anti-Smek1antibodies showed that a slower mobility band was induced by treatingcells with high osmolarity or UV irradiation (FIG. 3A, B). The effect ofpotato acid phosphatase treatment confirmed that the mobility shift wascaused by phosphorylation induced by sorbitol treatment (FIG. 3D).Further experiments showed that Smek1 phosphorylation could be inducedby various stress stimuli besides UV and sorbitol, including inhibitorsof protein synthesis, MMS, H₂O₂ and IL-1 (data not shown). However, itwas not triggered by the stimulation of the Fas death receptor usinganti-Fas antibody, or by certain DNA-damaging reagents includingetoposide, hydroxy urea and γ irradiation. In order to identify theprotein kinase(s) responsible for the stress-induced phosphorylation ofSmek1, various kinase inhibitors were tested, including SB203580, U0126,PD98059, wortmannin and rapamycin. However, none of these abolished thestress induced phosphorylation.

Example 3 Smek1 is a Substrate for p38 MAP Kinases

The stress-induced phosphorylation of Smek1 is sustained rather thantransient (FIG. 3C), which is similar to the kinetics ofstress-activated MAP kinases including the JNK and p38 MAP kinasefamilies (Chang and Karin 2001). To determine whether Smek1 can bephosphorylated by JNK MAP kinase, GST-Smek1 fusion protein was purifiedfrom bacteria and tested as a substrate in an in vitro kinase assay withimmunoprecipitated JNK. The results indicated that Smek1 was notphosphorylated by JNK in vitro, although the positive control GST-cJunwas strongly phosphorylated by JNK (FIG. 4A). Furthermore, thephosphorylation pattern of Smek1 was not altered in jnk1−/−jnk2−/−double knockout MEF cells (data not shown), suggesting that other stresskinases were involved. The lack of inhibition of Smek1 phosphorylationby SB203580, which blocks p38α and p38β activation, suggests thatneither are essential; however, the other two p38 isoforms, p38γ andp38δ, are insensitive to SB203580. To test whether p38γ and p38δ couldbe the kinases responsible for Smek1 phosphorylation, immunoprecipitatedp38 isoforms were analyzed in kinase assays using GST-Smek1 as asubstrate. The results from this study indicated that Smek1 wasdifferentially phosphorylated by p38 MAP kinases (FIG. 4B). It is clearthat p38δ had highest activity towards GST-Smek1 among the four p38isoforms, whereas p38γ showed modest activity. In contrast, p38α and βonly caused minor phosphorylation of GST-Smek1 compared to the positivecontrol GST-ATF2, which is consistent with the lack of effect ofSB203580 in vivo. To confirm that the observed phosphorylation ofGST-Smek1 was not due to a co-precipitated kinase, p38γ and p38δ mutantslacking kinase activity (p38γ-AF and p38δ-KM) were tested and asexpected, these mutant proteins did not cause detectable phosphorylationof GST-Smek1 (FIG. 4C).

The serine residues within the SP cluster of Smek1 are the majorphosphorylation sites by a stress-activated kinase in vivo. Based onvisual examination of Smek1 sequence, the cluster of five consecutive SPsites in the C-terminal region of Smek1 conform to the consensusphosphorylation sites for p38 MAP kinases. To prove these were the majorphosphorylation sites, a mutant Smek1 containing five Serine to Alaninemutations was created using site-directed mutagenesis. In the kinaseassay, GST fusion of the mutant protein (GST-Smek1-5A) showeddramatically decreased phosphorylation by p38δ and p38γ compared towildtype Smek1 (FIG. 4C). Furthermore, when FLAG-tagged Smek1-5A wasexpressed in mammalian cells, no mobility shift was observed in responseto various stress stimuli (FIG. 4D).

Example 4 Smek1 Interacts with FOXO Transcription Factors

Experiments depleting the single Smek1 homolog in C. elegans using RNAidemonstrated that the worm Smek1 homolog plays a role in regulatingstress resistance and organismal longevity (See Example 6). The similarphysiological role of Smek1 and FOXO homolog Daf16 suggested that theymight function within a complex. Flag-tagged Smek1 and HA-tagged FOXO3aor FOXO4 were co-expressed in 293T cells, and both FOXO3a and FOXO4 weredetected in immunoprecipitates containing Smek1 (FIG. 5A, lane 3 and 4).Similarly Smek1 was also detected in the immunoprecipitates of FOXO3a.To investigate the effect of stress-induced phosphorylation of Smek1 onSmek1-FOXO interaction, we co-expressed FOXO3a with wildtype Smek1 andmutant Smek1 (Smek1-5A), respectively, and analyzed their interactionsby co-immunoprecipitation and western blotting. The results from thisexperiment showed that Smek1 mutant lacking the phosphorylation siteshad significantly weaker affinity for FOXO3a (FIG. 5B, left panel),suggesting that stress signaling might promote Smek1-FOXO interaction.The difference in FOXO binding is not due to differences in expressionlevel or localization, as similar amounts of FOXO3a, Smek1 and Smek1/5Aproteins were detected in cell lysates and immunoprecipitates by westernblot analysis (FIG. 5B). In addition, Smek1-5A is still localized in thenucleus.

FOXO proteins are well-known downstream target for theinsulin/IGF-1-PI3K-Akt pathway. The phosphorylation of FOXOs by Akt hasbeen shown to cause cytoplasmic retention of FOXOs through 14-3-3binding, thereby inhibiting the transcriptional activity of FOXOs(Brunet, Bonni et al. 1999). To determine whether the phosphorylationvia Akt negatively regulates the interaction between Smek1 and FOXO,wildtype FOXO4 and a mutant FOXO4 lacking the three Akt phosphorylationsites (FOXO4-TM), were co-expressed with Smek1 in 293T cells forimmunoprecipitation analysis. The results indicated that FOXO4-TM boundSmek1 several times more strongly than wildtype FOXO4, although bothproteins were expressed at similar levels (FIG. 5A, lane 4 and 5). AsFOXO4-TM is constitutively nuclear, this observation raises thepossibility that Smek1 and FOXO might interact in the nucleus.

Example 5 Smek1 Promotes FOXO-Driven Transcription

Smek1 plays a role in regulating FOXO-driven transcription.Co-expression of Smek1 and FOXO3a with luciferase reporters for FOXOproteins in mammalian cells demonstrated the effects of Smek1 ontranscription by FOXO. First, the activity of FOXO3a towards a syntheticreporter containing three FOXO binding sites (pGL2-3xIRS-luc) in theabsence or presence of Smek1 in HepG2 hepatocytes was tested. The datashowed that Smek1 alone did not activate transcription of the syntheticreporter transcription. However, it promoted FOXO-driven transcriptionwhen co-expressed with either FOXO3a or FOXO3a-TM (FIG. 6A). Differentlevels of Smek1 protein were tested in the reporter assay to furtheranalyze the transcriptional activation by Smek1. The increase inFOXO-mediated transcription correlated with the amount of Smek1co-expressed with FOXO3a-TM (FIG. 6B). Co-expression of Smek1consistently resulted in robust activation of gene expression driven byFOXO3a mutant lacking the Akt phosphorylation sites, which is consistentwith its stronger interaction with Smek1.

Further, Smek1 acts on luciferase reporters driven by a native promoterof FOXO target genes. The reporters for two genes involved in thecellular protective response, GADD45 and catalase, were tested. Theresults showed that Smek1 by itself caused a modest activation ofpGADD45-luc reporter and a strong activation of catalase promoter, whichmight result from its activation of endogenous FOXO (FIG. 6C).Furthermore, co-expression of Smek1 and FOXO3a led to synergisticactivation of both reporter gene transcription (FIG. 6C). In the case ofcatalase reporter, the enhanced transcription is not observed with adominant negative FOXO3a mutant consisting of only the DNA bindingdomain, FOXO3a-DB (Dijkers, Birkenkamp et al. 2002) (FIG. 6D),suggesting that FOXO3a activation domain is required for the effect.

Example 6 C. elegans RNAi Depletion of the Worm Smek1 Homolog

In worms, the single Smek1 homolog is most highly conserved with thehuman Smek1. Both have a nuclear localization sequence at theC-terminus, an EVH1 domain required for protein interactions and aconserved domain, DUF625, whose function is unknown. Also conserved is ashort amino stretch at the C-terminus that resembles a DNA bindingdomain. Given the homology, several experiments involving depletion ofthe Smek1 homology in C. elegans using RNAi were conducted to furtherelucidate the role of Smek1.

To confirm the link between Smek1 in the regulation of daf-16 (the wormhomolog of FOXO), Smek1 was depleted, using RNAi, in long-liveddaf-2(e1370) mutant animals, completely suppressing the long life spanof the mutant animals when compared to non-mutant animals. Thisdemonstrated that Smek1 was an essential component of insulin/IGF-1signaling in the worm. In fact, lower Smek1 activity reduced the longlifespan of daf-2(e1370) mutant animals to the same extent as did RNAidirected towards daf-16. Furthermore, wild type animals treated withSmek1 RNAi demonstrated a moderate reduction of lifespan. This reducedlifespan was similar to daf-16 RNAi treated animals, or daf-16(mu86)null mutant animals.

To further confirm the link, Smek1 was depleted in daf-16(mu86) mutantanimals. As expected, unlike wild type animals, reduced Smek1 activitydid not reduce the lifespan of daf-16 null mutant animals.

Smek1 is specific to the insulin/IGF-1 signaling pathway. Because lowerSmek1 gene activity results in reduced longevity, demonstration ofspecificity to the insulin/IGF-1 signaling pathway requiresdemonstration that reduced Smek1 activity does not result in a generaldecline of longevity in all long-lived mutant animals. Besides theinsulin/IGF-1 pathway, RNAi or mutation of components of themitochondrial electron transport chain (ETC) increases longevity. Incontrast to the insulin/IGF-1 pathway, the ETC pathway is requiredduring larval development and does not depend upon daf-6 for increasedlongevity. Three ETC pathway mutants were tested with Smek1 depletion:cyc-1 RNAi (complex III component), isp-1(qm150) or clk-1(qm30) animals.In all three cases, Smek1 was not required for the long lifespan ofanimals with compromised complex III activity, cyc-1 RNAi treated,isp-1(qm150) mutant animals. Smek1 RNAi also did not suppress the longlifespan of clk-1 mutant animals, which have defects in mitochondrialubiquinone synthesis. Therefore, reduced Smek1 gene function does notcause a general sickness that results in reduced longevity.

Taken together, three pieces of data indicate that Smek1 is an essentialcomponent of the insulin/IGF-1 pathway to regulate the aging process inworms. One, reduced Smek1 activity completely suppresses the longlifespan of daf-2(e1370) mutant animals. Two, reduced Smek1 activity didnot further shorten the lifespan of daf-16(mu86) null mutant animals,but did decrease the longevity of wild type animals, much like daf-16mutations do. Three, much like daf-16, Smek1 is not required for thelong lifespan cause by altered mitochondrial activity.

Smek1 does not act by regulation of nuclear entry of daf-16 (the wormFOXO homolog). In wild-type animals, daf-16 is predominantly in thecytoplasm due to inhibitory phosphorylation of serine and threonineresidues by the akt and sgk kinases. However, in daf-2 long-lived mutantanimals, daf-16 accumulates in the nucleus due to the lack of inhibitoryphosphorylation. Using a complementing DAF-16::GFP fusion protein, wildtype animals treated with daf-2 RNAi readily accumulated DAF-16 withinnuclei, Animals treated with daf-2 and Smek1 RNAi simultaneously alsoaccumulated DAF-16 in the nuclei of many cells, similar to animalstreated with an equally diluted cocktail of daf-2 and control plasmidRNAi. Therefore, DAF-16 can still enter the nucleus of cells withreduced Smek1 activity in response to lower insulin/IGF-1 signalingdaf-2. However, the increased nuclear entry of DAF-16 in the absence ofSmek1 does not result an increased lifespan. Therefore, much likeresults of Lin et. al, nuclear entry of daf-16 is not sufficient toconfer increased longevity of worms.

Smek1 does play an important role in the transcriptional activation ofdaf-16 target genes. sod-3, a well characterized daf-16 regulated gene,required Smek1 activity for expression. In all cases, reduced Smek1activity abolished the normally robust sod-3::GFP reporter expression inresponse to lower daf-2 activity. In fact, reduced Smek1 activityresulted in comparable loss of SOD-3::GFP expression when compared toanimals with reduced daf-16 activity.

Collectively, the data obtained from depletion of Smek1 in C. elegansconfirmed that Smek1 is an essential co-factor of daf-16 (FOXO) and thecombined action of both Smek1 and daf-16 is required for the propertranscriptional activation of daf-16 target genes.

As discussed above, longevity and stress resistance are highlycorrelated as are longevity and the insulin/IGF-1 pathway. Smek1provides an essential link between the pathways as demonstrated above.Furthermore, additional RNAi depletion experiments further confirm thisessential role of Smek1. Wild type or daf-2(e1370) mutant animalstreated with Smek1 were not sensitive to heat stress indicating theSmek1 does not play a role in thermotolerance. Smek1 is, however,essential for other stress resistance pathways. For example, wild typeanimals or daf-2(e1370) mutant animals with reduced Smek1 activity weresensitive to UV and oxidative stresses, such as paraquat. Furthermore,Smek1 is required for innate immunity, since reduced Smek1 activityresulted in wild type or daf-2(e1370) mutant animals that were moresensitive to Pseudomonas Aeurogis infection compared to control animals.Therefore, it is interesting to note that daf-2(e1370) mutant animalstreated with Smek1 RNAi are resistant to heat stress, but are notlong-lived or resistance to oxidative stress or infection, indicatingthat the longevity conferred by lower insulin/IGF-1 signaling may dependmore on resistance to oxidation and infection, rather than heat.

Finally, the insulin/IGF-1 pathway in worms independently regulatesdauer development, reproductive timing and longevity. But Smek1 is notrequired for DAF-16's dauer development and reproduction functions.Reduced Smek1 activity did not alter dauer development or reproductivetiming. Wild-type animals treated with Smek1 RNAi did not enter dauerdiapuase at 25° C. and daf-2(e1370) mutant animals treated with Smek1RNAi arrested as dauers at 25° C. Additionally, daf-2(e1370) mutantanimals still paused for 24 hours during the L2 larval stage whentreated with Smek1 RNAI, but not when treated with daf-16 RNAI. Thusindicating that Smek1 does not play a role in dauer development.Further, reduced Smek1 activity in either wild type or daf-2(e1370)mutant animals did not affect reproduction. For example, wild typeanimals with reduced Smek1 activity reproduced at the same rate asanimals on control bacteria and daf-2(e1370) mutant animals had aprotracted reproductive schedule that was nearly identical todaf-2(1370) mutant animals treated with Smek1 RNAi. Thus, consistentwith previous studies, the insulin/IGF-1 pathway can be diverged toregulate the timing of reproduction independently of longevity. Takentogether, Smek1 appears to be a unique factor that is solely requiredfor DAF-16's longevity function and is not required for the dauerdevelopmental or reproductive functions of DAF-16. Thus, Smek appears tobe an ideal target for modulation for affecting longevity given that itsassociation with FOXO is necessary for longevity but not for FOXO'sother roles. Thus modulation of Smek1 is less likely to have negativeside effects than other genes known to be involved in longevity such asFOXO.

Example 7 Further Characterization of smk-1

Sequence and ontogenetic analysis links SMK-1 with cell cycleprogression and carbohydrate metabolism, two processes regulated byinsulin signaling and FOXO activity in mammals. RNAi against smk-1results in phenotypes that include embryonic lethality, slow growth, andprotruding vulvas, suggesting that smk-1, like daf->6, is important fordevelopment during the embryonic and reproductive stages of the wormlife cycle (Kamath et al., 2003; Simmer et al., 2003). smk-1 shares 74%amino acid homology with human and mouse SMEK-1 (FIG. 1).

Several functional motifs are conserved between SMK-1 and the mammalianSMEK-1, including an EVH1 domain at the N-terminus, a conserved domainof unknown function (DUF625) in the central region and a third conservedregion (CR3) near the C-terminus. SMK-1 additionally contains conservedLXXLL (LDALL) and LLXXL (LLSTL) motifs (LLINL and LLRTL in human andmouse SMEK-1). These motifs are used by mammalian transcriptionalco-activators, such as PGC-1α and p300/CBP, to bind to either PPAR-γ, anuclear hormone receptor, or the forkhead transcription factor, FOXO1(Puigserver et al., 2003; Puigserver and Spiegelman, 2003).

Example 8 smk-1 is Expressed in the Nuclei of Intestinal and NeuronalCells in Adult Worms

We examined the timing and localization of SMK-1 within wild-typeanimals. Using a gfp tagged smk-1 cDNA construct under the control ofthe endogenous smk-1 promoter to create a stable transgenic line, weobserved strong nuclear localization of SMK-1-GFP in intestinal cells.GFP fluorescence was also detected in the nuclei of several hypodermalcells, in many neurons in the head and tail, and in the intestinal cellsof developing larvae. The GFP signal was reduced upon treatment withsmk-1 RNAi with the most pronounce reduction in the intestinal cells.Endogenous SMK-1 could also be detected in the nuclei of intestinalcells by staining with affinity-purified SMK-1 antibodies. The timingand localization of SMK-1 expression in worms was consistent with theknown developmental phenotypes of smk-1 caused by RNAi treatment.Importantly, these assays indicated that SMK-1 was temporally andspatially co-localized with active DAF-16, which is active intranscribing genes when expressed in the nuclei of these cells (Libinaet al., 2003).

Example 9 smk-1 is Required for daf-16 Dependent Regulation of Longevity

In addition to its role in innate immunity (Garsin et al., 2003), daf-16regulates genes necessary for daf-2 dependent longevity in worms. UsingRNAi against smk-1, we tested whether smk-1, like daf-16, was requiredfor the extension of dof-2 mutant lifespan. Reduced levels of smk-1completely suppressed the extended longevity of daf-2(e1370) mutantanimals (FIG. 9A, Table 1). However, snik-1 RNAi only moderatelyshortened the lifespan of wild-type worms (FIG. 9B, Table 1). The levelof lifespan suppression in wild-typc animals treated with smk-1 RNAi wassimilar to the reduced life spans observed in daf-16 RNAi treatedanimals or in daf-16(mu86) null mutant animals (Dillin et al., 2002a;Lin et al., 2001) (FIG. 9E, Table 1).

Because reduced smk-1 gene activity suppressed the extended lifespan ofdaf-2 mutant animals, we tested whether smk-1 RNAi was actingspecifically on the insulin/IGF-1 pathway or whether it caused a generaldecline in longevity in all long-lived mutant animals. Mutation orreduced expression of components of the mitochondrial electron transportchain increases longevity independently of daf-16 activity (Dillin etal., 2002b; Feng, 2001; Lee et al., 2003b). smk-1 was tested todetermine whether smk-1 was required for the increased longevity ofisp-1(qm150), clk-1(qm30) mutants, or animals treated with cyc-1 RNAi(complex III component). We found that smk-1 RNAi only slightlysuppressed the extended lifespans of the animals with compromisedcomplex III activity, i.e., the cyc-1 RNAi-treated animals andisp-1(qm150) mutant animals (FIGS. 9C and 9D, respectively, and Table1). Additionally, smk-1 RNAi did not fully suppress the long lifespan ofclk-1(qm30) mutant animals (Table 1), which have defects inmitochondrial ubiquinone synthesis (Jonassen et al., 2001; Miyadera etal., 2001). In each of these experiments, smk-1 RNAi-treated animalslived as long or longer than the same animals treated with daf-16 RNAi.smk-1's dispensability for pathways that work independently of daf-16activity confirms that smk-1 RNAi does not cause a general sickness inlong-lived animals but rather specifically affects insulin/IGF-1signaling(IIS)-regulated lifespan.

To further define the role of smk-1 in IIS, smk-1 was tested todetermine whether the function of smk-1 was coincident with or separablefrom the requirements for daf-16 in DAF-2 pathway mediated longevity.Smk-1 was first tested to determine whether smk-1 reduced the lifespanof daf-16(mu86) mutant animals. Unlike its effects on wild-type animals,reduced smk-1 activity did not reduce the lifespan of daf-16 null mutantanimals (Table 1). This result indicated that the requirement for smk-1in the regulation of longevity in wild-type animals is coincident withthe requirement for daf-16.

The overlapping function of smk-1 with daf-16 in wild-type animalssuggests that smk-1 might be required for daf-16 dependent increases inlongevity mediated by other mechanisms. Because daf-16 is essential forthe extended lifespan observed in wild-type animals lacking a germline(Hsin and Kenyon, 1999), we asked whether genetically germline-ablatedanimals would show a reduction in lifespan when treated with smk-1 RNAi.Using glp-1(e2141) mutant animals that lack germline cells at thenon-permissive temperature (25° C.), we found that these long-livedmutant animals required smk-1 for their increased longevity (FIG. 9F,Table 1). smk-1 RNAi suppressed the long lifespan of glp-1 mutantanimals to the same extent as daf-16 RNAi. Together, these resultsindicate that smk-1 cannot act independently from daf-16 in wild-typeanimals and that smk-1 is required for both known forms of daf-16dependent longevity.

Taken together, four pieces of data indicate that smk-1 is an essentialcomponent of the insulin/IGF-1 pathway that regulates the aging processin worms: 1) Reduced smk-1 activity completely suppressed the longlifespan of daf-2(e1370) mutant animals; 2)) smk-1 is not required forthe long lifespan caused by altered mitochondrial activity; 3) Reducedsmk-1 activity did not further shorten the lifespan of daf-16(mu86) nullmutant animals, but did decrease longevity modestly in wild-typeanimals; 4) Reduced smk-1 activity completely suppressed the increasedlongevity due to loss of the germline.

Example 10 smk-1 is not a Transcriptional Target of DAF-16

One possible mechanism by which smk-1 could be required for DAF-16dependent longevity is that smk-1 is a transcriptional target of DAF-16.Recently, through microarray analysis, several transcriptional targetsof DAF-16 have been identified and found to be physiologically relevantfor DAF-16-mediated longevity (Murphy et al, 2003). smk-1 was examinedto determine whether it could be a transcriptional target of DAF-16required for longevity in worms using quantitative real time PCR(Q-PCR); wild-type worms treated with daf-16 RNAi did not exhibitreduced levels of smk-1 mRNA compared to worms treated with empty vectorRNAi. smk-1 mRNA levels were significantly diminished in worms treatedwith smk-1 RNAi, confirming the specificity and penetrance of the RNAiconstruct. Moreover, smk-1 and its mammalian homologue have not appearedas DAF-16/FOXO3a dependent genes in microarrays and screens identifyinggenes differentially regulated during the aging process (McCarroll etal., 2004; McElwee et al., 2004; Murphy et al., 2003). Additionally,despite the relative abundance of short consensus binding sites forDAF-16 within the complete C. elegans genome, no DAF-16 binding sitesare present within the smk-1 promoter (the 2.0 kb promoter regionupstream of the smk-1 coding sequence) or within the first intron ofsmk-1. Finally, fluorescence levels of our smk-1::gfp overexpressionlines did not appear visibly reduced upon treatment with daf-16 RNAi.These data suggest that smk-1 is not directly or indirectlytranscriptionally regulated by DAF-16.

Example 11 daf-16 is not a Transcriptional Target of SMK-1

A second mechanism by which SMK-1 might regulate daf-16 dependentlongevity is through regulation of daf-16 transcription or proteinlevels. Again, this possibility seemed unlikely because levels of daf-16observed using a daf-16::gfp fusion gene under control of the endogenousdaf-16 promoter were not diminished in animals treated with smk-1 RNAi,and western blot analysis indicated that the levels of DAF-16-GFP werenot diminished. Additionally, using quantitative PCR, no decrease indaf-16 mRNA levels in daf-2(e1370) animals treated with smk-1 RNAi wasobserved. Thus, smk-1 does not to appear to regulate daf-16transcription directly or to alter protein levels to a detectableextent. This again suggests that smk-1 must affect daf-16 dependentlongevity by another mechanism.

Example 12 Nuclear Entry of DAF-16 is Independent of SMK-1

In wild-type animals, DAF-16 is predominantly localized in the cytoplasmas a result of inhibitory phosphorylation of Ser/Thr residues by the AKTand SGK kinases. However, in long-lived daf-2 mutant animals, DAF-16accumulates in the nucleus due to a lack of inhibitory phosphorylationat these sites (Henderson and Johnson, 2001; Hertweck et al., 2004; Linet al., 2001). smk-1 was tested to determine whether SMK-1 was requiredfor the nuclear accumulation of DAF-16. Using a complementingdaf-16::gfp fusion gene (Henderson and Johnson, 2001), wild type animalstreated with daf-2 RNAi readily accumulated DAF-16-GFP protein withintheir nuclei, as monitored by the nuclear accumulation of the GFPfluorescence signal. As a negative control, animals treatedsimultaneously with both daf-2 and daf-16 RNAi had a diminished GFPsignal, presumably due to daf-16 RNAi acting on the daf-16::gfp fisiongene product. Interestingly, and in contrast to results obtained usingdaf-16 RNAi, animals treated simultaneously with daf-2 and smk-1 RNAIaccumulated DAF-16-GFP in nuclei to the same degree as animals treatedwith an equally diluted mixture of daf-2 and control RNAi plasmid. Thus,in response to decreased insulin/IGF-1 signaling, DAF-16 can still enterthe nucleus of cells that have reduced smk-1 activity. It is importantto note, however, that despite the nuclear accumulation of DAF-16, inthe absence of smk-1, nuclear localized DAF-16 did not result inincreased lifespan, supporting previous conclusions that nuclear entryof DAF-16 is not sufficient for increased longevity (Lin et al., 2001).

Example 13 Nuclear Entry of SMK-1 is Independent of DAF-16

Because nuclear entry of DAF-16 was not dependent upon smk-1, smk-1 wastested to determine whether nuclear entry of SMK-1 was dependent upondaf-16. Using the smk-1::gfp strain, treatment of animals with eitherdaf-16 or daf-2 RNAi did not alter nuclear accumulation of SMK-1-GFP asmeasured by fluorescence of the GFP. Therefore, SMK-1 nuclearlocalization is independent of DAF-16, and, unlike DAF-16, SMK-1 islocalized to the nucleus of intestinal cells regardless of IIS status.

The data from these four sets of experiments indicate that SMK-1 andDAF-16 do not appear to co-regulate expression or influence each other'snuclear entry, indicating that SMK-1 affects DAF-16 activity in someother manner. The nuclear localization of SMK-1 suggests that SMK-1could directly influence DAF-16 transcriptional activity.

Example 14 SMK-1 is Required for DAF-16 Transcriptional Activity

Based on the RNAi data, one would predict that loss of smk-1 shouldreduce transcription of DAF-16-dependent genes. Therefore, we askedwhether smk-1 RNAi could influence the mRNA levels of well-characterizedDAF-16 target genes. Using daf-2(e1370) mutant worms expressing anintegrated sod-3::gfp reporter construct, smk-1 RNAi reduced thenormally robust GFP reporter expression of this strain. These effectswere quantified using a fluorimeter to measure the levels of sod-3::gfpexpression in an entire population of worms. In the daf-2(e1370) mutantbackground, reduced smk-1 activity resulted in a decrease of sod-3::gfpexpression comparable to that seen in animals treated with daf-16 RNAi.In each case the reduction was approximately 20%. These results werealso confirmed using Q-PCR to analyze the endogenous sod-3 transcript ofdaf-2(e1370) animals treated with either daf-16 or smk-1 RNAi. In eachcase, the respective RNAi reduced the RNA expression by 60-70%.

FOXO3a and DAF-16 function as both transcriptional activators andrepressors (Jia et al., 2004) (Schmidt et al., 2002). SMK-1 was examinedto determine whether it was also required for the repressor activity ofDAF-16. Using Q-PCR, daf-15 was tested to determine whether it, a genethat is transcriptionally repressed by DAF-16 (Jia et al., 2004), wasalso repressed in the absence of smk-1. In daf-2(e1370) mutant animals,daf 15 expression was repressed by nuclear DAF-16. However, reduceddaf-16 resulted in upregulation of daf-15 transcripts more than 150% asdetermined by QPCR. In a similar manner, reduced smk-1 also resulted inincreased expression of daf-15 mRNA more than 50% as determined by QPCR,suggesting that SMK-1 is required for the transcriptional repressoractivity of DAF-16.

Human SMEK-1 was tested to determine whether it functioned as atranscriptional regulator of human FOXO proteins. Using a synthetic FOXOluciferase reporter containing three tandem IRS (insulin responsesequences) elements, increased levels of SMEK-1 enhanced transcriptionof these FOXO3a reporter genes in transient assays in 293 and HepG2cells. The enhanced transcription in 293 cells showed an increase inFOXO3a-mediated transcription that was dose dependent with a 12-foldincrease in expression at the highest levels of SMEK-1 supplied. Theenhanced transcription in the HepG2 cells was more robust with a triplephosphorylation mutant of FOXO3a (FOXO3a-TM) that is constitutivelynuclear and therefore hyperactive with the FOXO3a-TM cell line showingnearly double the activity when supplemented with SMEK-1 where theFOXO3a wild type cell line only showing a fifty percent increase withthe same amount of SMEK-1. Similar dose-dependent transcriptionalactivation was also observed with hyperactivated AFX (AFX-AAA), anotherFOXO homolog in mammalian cells. SMEK-1's ability to enhance expressionof a known FOXO target gene was examined by measuring transcriptionalactivity of the native GADD45 promoter. Overexpression of SMEK-1 aloneresulted in increased levels of GADD45 reporter activity that showeddose dependent increase in activity. Activation of the native promoterwas further enhanced by co-expression of both SMEK-1 and FOXO3a. Theenhancement of transcription due to SMEK-1 seems to requireco-expression of FOXO3a when synthetic FOXO reporters were used;however, SMEK-1 alone was sufficient to activate the native promoter, atleast partially. Finally, consistent with the repression of daf-15expression by SMK-1 in worms, a Gal4DB-SMEK-1 fusion protein showeddose-dependent capacity for repressor activity in a Gal4-luciferasereporter gene assay.

Taken together, the requirement for SMK-1 for the transcriptionalinduction of sod-3::gfp and the transcriptional repression of daf-15,and the evidence that mammalian SMEK-1 enhances the transcriptionalactivity of both synthetic and endogenous FOXO3a reporters in mammaliancell lines, support a model in which SMK-1 functions as atranscriptional cofactor for DAF— 16/FOXO3a.

Example 14 smk-1 Regulates Longevity Independent of Insulin/IGF-1'sRoles in Development and Reproduction

In worms the insulin/IGF-1 pathway independently regulates dauerdevelopment, reproductive timing and longevity (Dillin et al., 2002a).Because smk-1 is required for daf-16-dependent longevity, smk-1 wastested to determine whether it was also required for daf-16 to regulatethe dauer development and reproductive functions. In fact, reduced smk-1activity did not alter dauer development or reproductive timing. Whilewild-type animals treated with smk-1 RNAi did not enter dauer diapauseat 25° C., daf-2(e1370) mutant animals treated with smk-1 R(NAI arrestedas dauers at 25° C. Additionally, daf-2(e1370) mutant animals paused for24 hr during the L2 larval stage at the permissive temperature whentreated with smk-1 RNAi but not when treated with daf-16 RNAi. Theseresults indicate that smk-1 does not play a role in dauer larvaldevelopment.

In addition, reduced smk-1 activity in either wild-type or daf-2(e1370)mutant animals did not affect reproduction. For example, wild-typeanimals treated with smk-1 RNAi reproduced at the same rate as animalson control bacteria, and daf-2(e1370) mutant animals had a protractedreproductive schedule that was nearly identical to daf-2(1370) mutantanimals treated with smk-1 RNAi. Thus, consistent with previous studies,the insulin/IGF-1 pathway can diverge to regulate the timing ofreproduction independently of longevity (Dillin et al., 2002a). SMK-1 isnot required for daf-2 dependent entry into dauer or daf-2 dependentextension of reproduction. Thus, SMK-1 appears to be unique in being afactor that is solely required for the longevity function of DAF-16.

Example 15 Generation of Smek Plasmids and Antibodies

HA-tagged FOXO3a, FOXO4 (AFX) and FOXO4-TM expression constructs werekindly provided by K. Arden (UCSD). pECE/FOXO3a and pECEiFOXO3a-TM weregifts from M. Greenberg (Harvard). FLAG-tagged p38 MAPK plasmids andGST-ATF2 plasmid were gifts from J. Han (Scripps). pGAD45-luc reporterwas a gift from N. Motoyama (National Institute for Longevity Sciences,Japan). Human Catalase-luc plasmid was a gift from T. Finkel (NIH). cDNAfor human Smek1 splice form Smek1-S1 was provided by S. Sugano(University of Tokyo). The C-terminal sequences of Smek1 full-lengthwere constructed by subcloning inserts from two EST clones A1638670(SphI/EcoRV) and BG676909 (EcoRV/SacI) (The I.M.A.G.E. Consortium) intoSp72 vector cut with SphI and SacI. Plasmid for GST-Smek1 wasconstructed by joining a PCR fragment of Smek1-S1 (XbaI/SphI) with Smek1C-terminal fragment (SphI/XhoI). GFP-Smek-S1 and GFP-Smek1 plasmids werecreated by PCR amplification and subcloning of the corresponding Smek1fragments (KpnI/XhoI) into pEGFPN1 (KpnI/XhoI) (Clontech). FLAG-taggedSmek1 constructs were made by PCR and subeloning of Smek1 sequence(SpeI/NotI) into a modified pEGFPN1 plasmid containing three copies ofFLAG tag sequence but missing the GFP sequence (pFLA3). Smek1-5A mutantwas created using site-directed mutagenesis and subcloned into pGexKGand pFLA3 vectors, respectively. Anti-Smek1 antibodies were raised inrabbits immunized with a synthetic peptide of human Smek1.

Example 16 Cell Culture

HeLa cells, 293 cells, 293T cells and HepG2 cells were cultured in DMEMsupplemented with 10% fetal calf serum and the antibiotics penicillinand streptomycin at 37° C. 293 cells and 293T cells were transfectedusing the calcium phosphate method or the Effectene reagents (Qiagen).HepG2 cells were transfected using the FuGene 6 reagent (Roche).

A. Microscopy

Cells transfected with GFP-tagged constructs were fixed in 3.7%formaldehyde for microcopy analysis using the Deltavision deconvolutionmicroscope. For immunofluorescence staining, untransfected cells werefixed and permeablized in PBS containing 0.2% Triton X-100, blocked withnormal goat serum and stained with antibodies. Cell nuclei werevisualized by staining with Hoescht dye.

B. Immunoblotting and Immunoprecipitation

For western blotting and immunoprecipitation, cells were lysed in RIPAbuffer without SDS in the presence of protease inhibitors. The proteinconcentration of cell lysates was determined using the Bio-Rad DCProtein Assay kit. Lysates were either mixed with an equal volume of 2×sample buffer and boiled for 5 min or subjected to immunoprecipitation.The phosphatase treatment of immunoprecipitated Smek1 was describedpreviously (Meisenhelder, Suh et al. 1989).

For co-immunoprecipitation studies, cells were extracted in NP40 lysisbuffer (20 mM Hepes, pH 7.4, 2 mM EDTA, 2 mM EGTA, 100 mM NaCl, 50 mMNaF, 1 mM Na₃VO4, 1% NP40) plus protease inhibitors, clarified bycentrifugation at 15,000×g at 4° C. for 10 min, incubated with anti-FLAGantibodies immobilized on protein A Sepharose beads for 2-4 hrs at 4°C., wash four times with lysis buffer plus protease inhibitors, andresuspended in equal volume of 2× sample buffer for western blotanalysis.

Example 17 Kinase Assays

Activated JNK MAPKs were precipitated from 293T cells using anti-JNKantibodies and assayed using GST fusion proteins purified from BL21strain as substrates as described elsewhere (Perlman, Schiemann et al.2001). Activated FLAG-tagged p38 MAPKs were precipitated from 293T cellsusing anti-FLAG antibodies and assayed according to method describedpreviously (Jiang, Chen et al. 1996).

Example 18 Luciferase Assays

HepG2 cells from one 50-70% confluent 10 cm dish were split into one12-well plate for transfection. 293 cells and 293T cells were seeded in12-well plates at a density of 5×10⁴ cells/well. Cells were transfectedwith Luciferase reporter construct, H-Ras-LacZ construct together withvarious combinations of Smek1, FOXO3a plasmids. Two days after thetransfection, cells were lysed in 100 μl of lysis buffer and one fifthof the lysates were used in luciferase assay according to the Promegaprotocol. β-galactosidase activity was assayed as described elsewhere(Conkright, Canettieri et al. 2003).

Example 19 C. elegans Methods and Generation of Transgenic Lines

CF1037: daf-16(mu86)I, CF1041: daf-2(e1370)III, CB4037: glp-1(e2141)III, MQ887: isp-1(qm150)V, MQ167: clk-1(qm30)IV, CF1580:daf-2(e1370)III; muls84{pAD76(sod-3:.gfp)} (Libina et al., 2003),CF1553: muls84{pAD76(sod-3::gfp)} (Libina et al., 2003). Wild-type C.elegans (N2) strains were obtained from the Caenorhaditis GeneticsCenter. Nematodes were handled using standard methods (Brenner, 1974).For generation of AD24, AD25, and AD26 transgenic animals, plasmid DNAcontaining the pAD187 (smk-1::gfp) construct was mixed at 18 μg/ml with20 μg/ml of pRF4(rol-6) construct (Mello et al., 1991). Worms used ascontrols in lifespans against smk-1 overexpressing strains contained 75μg/ml of pRF4(rol-6) injected with 75 μg/ml of pAD158 (ges-1::gfp).Mixtures were microinjected into the gonads of adult hermaphroditeanimals by using standard methods (Mello et al., 1991). Transgenic F1progeny were selected on the basis of roller phenotype. Individualtransgenic F2 animals were picked to establish independent lines.

Example 20 Lifespan Analysis

Lifespan analyses were performed as described previously (Dillin et al.,2002). Eggs from strains grown at 20° C. degrees were transferred toplates seeded with RNAi bacteria (BL21-DE3). Adult animals were scoredevery other day for viability. Animals were judged as dead when theyceased pharyngeal pumping and did not respond to prodding with aplatinum wire at least three times. During their reproductive period,animals were transferred to new plates every other day. At the end oftheir reproductive period, animals were transferred to new plates atleast once per week. The pre-fertile period of adulthood was used as t=0for lifespan analysis. Strains were grown at 20° C. at optimal growthconditions for at least two generations before use in lifespan analysis.All lifespan analysis were conducted at 20° C. unless otherwise stated,Statview 5.01 (SAS) software was used for statistical analysis and todetermine means and percentiles. In all cases, P values were calculatedusing the log-rank (Mantel-Cox) method.

Example 21 Dauer Formation Assays

Eggs from daf-2(e1370) reproductive animals were transferred to platesseeded with RNAi bacteria and shifted to 25° C. for three days. Dauerformation was determined based upon morphology using a dissectingmicroscope. Percentage dauer formation was determined relative to emptyvector and daf-16 RNAi treated animals.

Example 22 Reproductive Assays

N2 eggs were incubated at 20° C. on plates seeded with various RNAitreatments. Worms were synchronized within one hour at the L1 stage uponhatching. Late L4 stage worms were picked and transferred to fresh RNAIplates every 12 hours for 4-5 days. After this period, the worms weretransferred every 24 hr. All plates were then incubated at 20° C. forabout 2 days and shifted to 4° C. The number of worms that developed wasdetermined at the end of the experiment. For RNAi treatments thatresulted in embryonic lethality, eggs were counted instead of hatchedprogeny.

Example 23 RNA Isolation and Quantitative RT-PCR

Total RNA was isolated from synchronized populations of approximately50,000 prefertile or day 1 reproductive animals. Animals were removedfrom plates and washed two times with M9 buffer followed by one time inDEPC water. Total RNA was extracted using TRIzol reagent (Gibco). cDNAwas created from 6 μg of RNA added to 2× reaction buffer usingSuperscript II RT (Invitrogen). SybrGreen real time qPCR experimentswere performed as described in the manual using ABI Prism7900HT (AppliedBiosystems). Primers and probes are listed below:

Primers:

act-1 forward GAGCACGGTATCGTCACCAA (SEQ ID NO 12) act-1 reverseTGTCATGCCAGATCTTCTCCAT (SEQ ID NO 13) sod-3 forwardCTAAGGATGGTGGAGAACCTTCA (SEQ ID NO 14) sod-3 reverseCGCGCTTAATAGTGTCCATCAG (SEQ ID NO 15) smk-1a forwardACCAACAGAGATCATATTCTTGACCAT (SEQ ID NO 16) smk-1a reverseGGTTGCGTCTCGTTTTATATCAAGAT (SEQ ID NO 17) daf-16a forwardGGAAGAACTCGATCCGTCACA (SEQ ID NO 18) daf-16a reverseTTCGCATGAAACGAGAATGAAG (SEQ ID NO 19) daf-15 forwardGCAATGTGTTCCCGTTTTTAGTG (SEQ ID NO 20) daf-15 reverseTAAGTCAGCACATGTTCGAAGTCAA (SEQ ID NO 21)

Example 24 GFP Localization

Paralyzed day one reproductive adult transgenic animals were assayed forGFP expression at 10× or 63× magnification using a Leica 6000B digitalmicroscope. When comparing fluorescence between samples ofdifferentially RNAi treated animals, only non-saturating pictures usingfixed times of exposure were taken. Images were acquired using LeicaFW4000 software.

Example 25 Fluorimetry

Eggs from daf-2 (e1370);sod-3:gfp reproductive animals were transferredto plates seeded with RNAi bacteria or empty vector controls. Eggstreated with daf-16 RNAi were transferred one day later to compensatefor developmental delays seen in daf-2 mutant strains. Upon day one ofadulthood, three populations of forty worms for each treatment werepicked and placed in wells containing M9 buffer. As a control,populations of day one adults were picked from N2 worms that did notcontain GFP expressing constructs. All measures of fluorescence occurredimmediately after transfer.

Fluorescence was measured using the HTS 7000 Plus BioAssay Reader at afixed gain of 110. Fluorescence was determined for each population intriplicate after shaking of the well to redistribute the worms.Fluorescence was measured using a six spot check. Levels of fluorescencewere normalized to background levels seen in the non-fluorescent strain.The experiment was repeated at least three times using independentlygrown populations of worms.

Example 26 RNAi Constructs

RNAi treated strains were fed E. coli (HT115) containing an emptycontrol vector pAD12 (Dillin et al., 2002a) or E. coli expressingdouble-stranded RNAi against the genes daf-16 (pAD43, Dillin et al.,2002a), daf-2 (pAD48, Dillin et al., 2002a), smk-1 (from the AhringerRNAI library, Simmer et al., 2003) or cyc-1 (from the Ahringer RNAilibrary, Simmer et al., 2003).

Example 27 Creation of smk-1::gfp Constructs

To construct the plasmid expressing SMK-1-GFP driven by smk-1 endogenouspromoter, sequences 3 kb upstream of smk-1 coding region were amplifiedfrom genomic DNA by PCR and inserted upstream of GFP sequences in theworm expression vector pAD1. Full-length smk-1 cDNA was amplified as N′-and C′-fragments from a first strand worm cDNA library by PCR. The N′fragment was digested with NotI and BglI, and the C′ fragment wasdigested with BglII and KpnI, respectively. Both fragments were ligatedand inserted downstream of the promoter sequences in-frame with the GFPsequence at the C-terminus. Primers for N′ fragment:Forward-GTTTTGCGGCCGCATG TCGGACACAAAAGAGGTATC (SEQ ID NO 22),Reverse-AGTGCCAGATCTC(GCCGACG (SEQ ID NO 23). Primers for C′ fragment:Forward-TGCTGCCCTCCCGGCATCTC (SEQ ID NO 24),Reverse-GTTTTGGTACCCTGGCCTGCGAAACTGTGGC (SEQ ID NO 25).

Example 28 Creation and Affinity Purification of SMK-1 Antibody

A rabbit polyclonal antiserum against worm SMK-1 was generated using aGST-fusion protein containing the last C-terminal 114 amino acidresidues of SMK-1. To affinity purify the SMK-1 antibody, rabbitanti-SMK-1 serum was incubated overnight at 4° C. with the correspondingantigen immobilized on PVDF membrane and eluted with 100 mM glycine (pH2.5) followed by neutralization with Tris (pH 8.4).

Example 29 Immunofluorescence Microscopy

Briefly, worms were pre-fixed with 3% paraformaldehyde for 15 min,followed by freeze and crack treatment on poly-L-lysine coated ringslides. After blocking non-specific staining by incubating worms inTRIS-buffered saline (TBS) containing 5% BSA (TBSB), worms wereincubated with affinity purified anti-SMK-1 antibodies overnight at 4°C., rinsed with TBSB and subsequently incubated with goat anti-rabbitFLAX 568 in TBSB. After gentle washing, samples were mounted inGEL/MOUNT (Biomeda) for immunofluorescence microscopy.

REFERENCES

The following references are hereby incorporated by reference in theirentirety.

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TABLE 1 Effects of smk-1 RNAi on lifespan and brood size. Mean Lifespan± 75^(th) Average (Total s.e.m. Percentile* Brood #Animals Treatment(days) P† (days) Size ± SD^(Δ) Died/Total)^(§) daf-2(e1370) mutant worms20° C. Vector (control) 48.2 ± 1.2 56 49/64 daf-16 RNAi 24.6 ± 0.6<0.0001 ‡ 27 45/65 smk-1 RNAi 26.6 ± 1.5 <0.0001 ‡, 34 57/64  0.0528^(a)glp-1(e2141) mutant worms 25° C. Vector (control) 22.1 ± 0.9 28 N.D.74/80 daf-16 RNAi 11.5 ± 0.3 <0.0001 ‡ 14 N.D. 76/86 smk-1 RNAi 11.7 ±0.3 <0.0001 ‡, 14 N.D. 67/81  0.5459^(a) isp-1(qm150) mutant worms 20°C. Vector (control) 32.8 ± 1.8 40 N.D. 24/55 daf-16 RNAi 20.1 ± 0.9<0.0001 ‡ 24 N.D. 42/79 smk-1 RNAi 26.1 ± 1.0  0.0001 ‡, 31 N.D. 31/76<0.0001 ‡ N2 20° C. Vector (control) 17.5 ± 0.5 20 46/78 cyc-1 RNAi 32.9± 1.4 <0.0001 ‡ 44 N.D. 51/80 (Complex III) cyc-1 & daf-16 25.7 ± 1.1<0.0001^(b) 33 N.D. 60/78 RNAi cyc-1 & smk-1 25.6 ± 0.9 <0.0001^(b), 30N.D. 65/79 RNAi  0.6683^(c) daf-2 RNAi 35.8 ± 1.9 48 N.D. 56/79 daf-2 &cyc-1 45.0 ± 2.0 <0.0001^(d) 60 N.D. 71/80 RNAi smk-1 RNAi 14.5 ± 0.4<0.0001 ‡ 16 70/79 clk-1(qm30) mutant worms 20° C. Vector (control) 19.3± 1.1 24 N.D. 66/80 daf-16 RNAi 15.5 ± 0.7  0.0058 ‡ 17 N.D. 55/79 smk-1RNAi 16.6 ± 0.7  0.1405 ‡, 17 N.D. 50/80  0.1768^(a) daf-16(mu86) mutantworms 20° C. Vector (control) 10.8 ± 0.4 14 N.D. 53/80 smk-1 RNAi 10.6 ±0.3  0.3810 ‡ 11 N.D. 61/80 *The 75^(th) percentile is the age when thefraction of animals alive reaches 0.25. †P values were calculated forindividual experiments, each consisting of control and experimentalanimals examined at the same time. ^(§)The total number of observationsequals the number of animals that died plus the number censored. Animalsthat crawled off the plate, exploded or bagged were censored at the timeof the event. Control and experimental animals were cultured in paralleland transferred to fresh plates at the same time. The logrank(Mantel-Cox) test was for statistical analysis. ^(Δ)Average brood sizewas calculated from the total brood size of at least 15 animals culturedindependently in each trial. ‡ Compared with worms grown on HT115bacteria harboring the RNAi plasmid vector which were analyzed at thesame time. ^(a)Compared to worms cultured continuously on HT115 bacteriaharboring the daf-16 RNAi plasmid Egg (□, at 20° C., which were analyzedat the same time. ^(b)Compared to worms cultured continuously on HT115bacteria harboring the cyc-1 RNAi plasmid which were analyzed at thesame time. ^(c)Compared to worms cultured continuously on mixed culturesof HT115 bacteria harboring the cyc-1 and daf-16 RNAi plasmid which wereanalyzed at the same time. ^(d)Compared to worms cultured continuouslyon HT115 bacteria harboring the daf-2 RNAi plasmid which were analyzedat the same time.

1. A method of identifying a compound that binds to a Suppressor of MEKnull polypeptide (Smek protein) comprising: contacting the Smek proteinwith a compound; and measuring binding between the compound and the Smekprotein.
 2. The method of claim 1 wherein the Smek protein is SEQ IDNO:1.
 3. The method of claim 1 wherein the Smek protein is SEQ ID NO:2.4. The method of claim 1 wherein the Smek protein is 90% identical toSEQ ID NO:1 wherein said Smek protein has affinity for FOXOtranscription factors.
 5. The method of claim 1 wherein the Smek proteinis 90% identical to SEQ ID NO:2 wherein said Smek protein has affinityfor FOXO transcription factors.